Methods of reducing immunogenicity against factor VIII in individuals undergoing factor VIII therapy

ABSTRACT

The present disclosure provides methods of administering chimeric and hybrid Factor VIII (FVIII) polypeptides comprising FVIII and Fc to subjects at risk of developing inhibitory FVIII immune responses, including anti-FVIII antibodies and/or cell-mediated immunity. The administration is sufficient to promote coagulation and to induce immune tolerance to FVIII. The chimeric polypeptide can comprise full-length FVIII or a FVIII polypeptide containing a deletion, e.g., a full or partial deletion of the B domain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationNumber PCT/US2013/021332, filed Jan. 12, 2013, which claims the benefitof U.S. Provisional Application No. 61/668,961, filed Jul. 6, 2012, andU.S. Provisional Application No. 61/586,103, filed Jan. 12, 2012, whichare incorporated by reference herein.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to the field of therapeuticsfor hemostatic disorders.

BACKGROUND ART

Hemophilia A is an X-linked bleeding disorder caused by mutations and/ordeletions in the Factor VIII (FVIII) gene resulting in a deficiency ofFVIII activity (Peyvandi, F. et al. Haemophilia 12:82-89 (2006). Thedisease is characterized by spontaneous hemorrhage and excessivebleeding after trauma. Over time, the repeated bleeding into muscles andjoints, which often begins in early childhood, results in hemophilicarthropathy and irreversible joint damage. This damage is progressiveand can lead to severely limited mobility of joints, muscle atrophy andchronic pain (Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96(2003), which is herein incorporated by reference in its entirety).

The A2 domain is necessary for the procoagulant activity of the FVIIImolecule. Studies show that porcine FVIII has six-fold greaterprocoagulant activity than human FVIII (Lollar & Parker, J. Biol. Chem.266:12481-12486 (1991)), and that the difference in coagulant activitybetween human and porcine FVIII appears to be based on a difference inamino acid sequence between one or more residues in the human andporcine A2 domains (Lollar, P., et al., J. Biol. Chem. 267:23652-23657(1992)), incorporated herein by reference in its entirety.

Treatment of hemophilia A is by replacement therapy targetingrestoration of FVIII activity to 1 to 5% of normal levels to preventspontaneous bleeding (Mannucci, P. M., et al., N. Engl. J. Med.344:1773-1779 (2001), which is herein incorporated by reference in itsentirety). e.g.

Plasma-derived FVIII (pdFVIII) and recombinant human FVIII (rFVIII)products are utilized for treatment (on-demand therapy) and prevention(prophylaxis therapy) of bleeding episodes. rFVIII was developed toreduce the risk of blood-borne pathogen transmission following thewidespread contamination of plasma products with HIV and hepatitisviruses, and to secure an adequate supply of FVIII product. However,hemostatic protection with current FVIII products is temporally limiteddue to a short half-life (t_(1/2)) of approximately 8-12 hours,requiring prophylactic injections three times per week or every otherday for most patients in order to maintain FVIII levels above 1%, alevel that has been established as protective against most spontaneousbleeding episodes. Manco-Johnson et al., New Engl J Med. 357(6):535-44(2007).

Many studies have shown that, even at high doses, on-demand therapy isnot effective in preventing arthropathy. Aledort L. et al., J InternMed. 236:391-399 (1994); Petrini P. et al., Am J Pediatr Hematol Oncol.13:280-287 (1991). The benefits of prophylactic therapy have beendemonstrated in numerous clinical studies. Aznar J. et al., Haemophilia6(3):170-176 (2000), Feldman B. et al., J Thromb Haemost. 4:1228-1236(2006), Kreuz W. et al., Haemophilia 4:413-417 (1998), Liesner R. etal., B J Haem. 92:973-978 (1996), Ljung R., Haemophilia. 4(4):409-412(1998), Löfquist T, et al., J Intern Med 241:395-400 (1997), Nilsson I,et al., B. J Int Med 232:25-32 (1992), Risebrough N. et al.,Haemophilia. 14:743-752 (2008), Van Den Berg H. et al., Haemophilia 9(Suppl. 1):27-31 (2003), Van Den Berg H. et al., Haematologica89(6):645-650 (2004) and Manco-Johnson et al., supra, established thatchildren started on primary prophylaxis after their first joint bleedhad significantly fewer bleeds and less joint damage than childrentreated on-demand.

Compared to on-demand treatment, prophylactic therapy also decreasesdisability, hospitalization rate, and time lost from school or work;Aznar J. et al., Haemophilia 6(3):170-176 (2000), Molho P. et al.,Haemophilia 6(1):23-32 (2000) and improves quality of life for patientsand their families. Coppola A. et al., Blood Transfus. 6(2): 4-11(2008). However, prophylactic therapy often requires use of centralvenous access devices in children, and their attendant risks ofinfection, sepsis, and thrombosis. In addition, despite the benefits,acceptance of and compliance with prophylaxis decreases with age, inpart because of inconvenience and invasiveness. Geraghty S. et al.,Haemophilia 12:75-81 (2006), Hacker M. et al., Haemophilia 7(4):392-396(2001). Thus, an rFVIII product with a prolonged plasma t_(1/2) wouldpotentially be of benefit. Lillicrap D., Current Opinion in Hematology17:393-397 (2010).

Reduced mortality, prevention of joint damage, and improved quality oflife have been important achievements due to the development of pdFVIIIand rFVIII. Prolonged protection from bleeding would represent anotherkey advancement in the treatment of hemophilia A patients. However, todate, no products that allow for prolonged hemostatic protection havebeen developed. Therefore, there remains a need for improved methods oftreating hemophilia due to FVIII deficiency that are more tolerable,longer lasting, and more effective than current therapies.

In addition, 15-30% of previously untreated patients developneutralizing anti-FVIII antibodies (inhibitors) after transfusion withFVIII products. Various techniques for avoiding such immune responseshave been considered. These techniques include high-dose toleranceprotocols, use of peptide decoys mimicking the anti-FVIII antibody,bypassing immune recognition with human/porcine FVIII hybrid molecules,neutralizing FVIII-reactive CD4 T-cells with anticlonotypic antibodies,using universal CD4 epitopes, and blocking costimulation of CTLA-4-Ig oranti-CD40L. See, e.g., Lei et al., Transfusion Medicine 105: 4865-4870(2005). Presentation of FVIII by immune cells in order to inducetolerance has also been studied. For example, Lei et al. found thatpresentation of FVIII domains on an Ig backbone in B cells prevented ordecreased antibodies. Id. In addition, Qadura et al. found thattolerogenic presentation of FVIII using immature dendritic cells mayreduce immunogenicity. Journal of Thrombosis and Haemostatis 6:2095-2104 (2008). However, such methods are costly, complicated (e.g.,by requiring co-administration of other therapeutics in combination withFVIII or administration of whole cells instead of relatively simpleproteins), likely to result in unwanted side-effects, and/orinefficient. Accordingly, there remains a need for simple methods oftreating hemophilia due to FVIII deficiency that are effective inpatients that develop inhibitory responses.

BRIEF SUMMARY

The present disclosure provides methods of administering Factor VIII(FVIII) that improve immune tolerance. The methods compriseadministering a chimeric polypeptide comprising a FVIII portion and anFc portion (rFVIIIFc) to a subject at risk of developing an inhibitoryFVIII immune response. In some embodiments, the subject would develop aninhibitory immune response if administered an equivalent dose of apolypeptide consisting of the FVIII portion. In some embodiments, thesubject has developed an inhibitory immune response or an inhibitoryFVIII immune response. The administration of a chimeric polypeptidecomprising a FVIII portion and an Fc portion can be sufficient to treata bleeding condition and to induce immune tolerance to FVIII. Theadministration can be prophylactic. In some embodiments, theadministration decreases the incidence of spontaneous bleeding orprevents bleeding.

The immune response can comprise inhibitory anti-FVIII antibodies. Theantibody titer is at least 0.6 Bethesda Units (BU), at least 1.0 BU, orat least 5.0 BU. The immune response can also comprise a cell-mediatedimmune response, for example, the release of a cytokine. The cytokinecan be IL-12, IL-4, or TNF-α, for example. The immune response can alsoresult in clinical symptoms such as increased bleeding tendency, highFVIII consumption, lack of response to FVIII therapy, decreased efficacyof FVIII therapy, and/or shortened half-life of FVIII.

Subjects at risk of developing an inhibitory immune response includethose with a mutation, deletion, or rearrangement in a FVIII gene. Insome embodiments, the subject does not produce a FVIII protein. In someembodiments, the subject has severe hemophilia. In some embodiments, thesubject has a relative (e.g., a parent, cousin, aunt, uncle,grandparent, child, or grandchild) that has developed an inhibitoryimmune response to FVIII or another therapeutic protein. In someembodiments, the subject is concurrently or was previously receiving atherapy that increases immune function when the FVIII is administered.In some embodiments the subject is receiving interferon therapy oranti-viral therapy in combination with FVIII. In some embodiments, thesubject at risk of developing an inhibitory immune response has agenetic polymorphism associated with an increased cytokine level, suchas increased TNF-α or increased IL10. In some embodiments, the subjectat risk of developing an inhibitory immune response has a TNF-α-308G>Apolymorphism or allele 134 of the IL10G microsatellite.

In some embodiments, the subject at risk for developing an inhibitoryFVIII immune response has not been previously exposed to FVIII. In someembodiments, the subject at risk for developing an inhibitory FVIIIimmune response has been exposed to FVIII. In some embodiments, thesubject at risk for developing an inhibitory FVIII immune response hashad less than 150, less than 50, or less than 20 FVIII exposure days.

In some embodiments, the subject at risk for developing an inhibitoryFVIII immune response has not previously developed an immune response toFVIII or another therapeutic protein. In some embodiments, the subjectat risk for developing an inhibitory FVIII immune response haspreviously developed an immune response to FVIII (pdFVIII or rFVIII) oranother therapeutic protein. In some embodiments, the subject at riskfor developing an inhibitory FVIII immune response has previouslydeveloped an immune response to a FVIII product such as ADVATE®,RECOMBINATE®, KOGENATE FS®, HELIXATE FS®, XYNTHA®/REFACTO AB®,HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®, ALPHANATE® KOATE-DVI®,or HYATE:C®. In some embodiments, the FVIII products is a full lengthFVIII, a mature FVIII, or a B-domain deleted FVIII.

The methods of administering FVIII provided herein can induce immunetolerance. In some embodiments, the administration reduces the number ofanti-FVIII antibodies in the subject, the titer of anti-FVIII antibodiesin the subject and/or the level of a cytokine (e.g., IL-12, IL-4, orTNF) in the subject compared to the number, titer, or level prior toadministration. In some embodiments, the administration reduces thenumber of anti-FVIII antibodies in the subject, the titer of anti-FVIIIantibodies in the subject and/or the level of a cytokine (e.g., IL-12,IL-4, or a TNF) in the subject compared to the number, titer, or levelthat resulted from a previous treatment with a polypeptide consisting ofa FVIII polypeptide. In some embodiments, the administration reduces thenumber of anti-FVIII antibodies in the subject, the titer of anti-FVIIIantibodies in the subject and/or the level of a cytokine (e.g., IL-12,IL-4, or TNF) in the subject compared to the number, titer, or levelthat would result from administration of polypeptide consisting of aFVIII polypeptide to the subject.

The methods comprise administration of a chimeric polypeptide comprisinga FVIII portion and an Fc portion. The FVIII portion can be human FVIII,full-length FVIII, or FVIII containing a full or partial deletion of theB domain. The FVIII portion can be a biologically active polypeptide,e.g., a FVIII polypeptide with coagulation activity. The FVIII portioncan be at least 90% identical, 95% identical, or identical to a FVIIIamino acid sequence shown in TABLE 2 without a signal sequence (aminoacids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6).The FVIII portion can also be at least 90% identical, 95% identical, oridentical to a FVIII amino acid sequence shown in TABLE 2 with a signalsequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351of SEQ ID NO:6). The Fc portion can be identical to the Fc amino acidsequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 oramino acids 2352 to 2578 of SEQ ID NO:6).

The chimeric polypeptide can be in a form of a hybrid comprising asecond polypeptide in association with the chimeric polypeptide. Thesecond polypeptide can consist essentially of or consist of an Fc.

In some embodiments, the methods comprise administration of a chimericpolypeptide at a particular dose. The dose can be, for example, a doseof 10-100 IU/kg, a dose of 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, or 90-100 IU/kg, or a dose of 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg.

In some embodiments, the chimeric polypeptide is administered to asubject with a bleeding condition. The bleeding condition can be, forexample, a bleeding coagulation disorder, hemarthrosis, muscle bleed,oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis, gastrointestinal bleeding, intracranialhemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bonefracture, central nervous system bleeding, bleeding in theretropharyngeal space, bleeding in the retroperitoneal space, orbleeding in the illiopsoas sheath. In some embodiments, the bleedingcoagulation disorder is hemophilia A.

The present disclosure also provides a method of administering aclotting factor to a subject at risk of developing an inhibitory immuneresponse to the clotting factor comprising administering to the subjecta chimeric polypeptide comprising a clotting factor portion and an Fcportion. Also provided is a method of inducing immune tolerance to aclotting factor in a subject, wherein the subject is a fetus, the methodcomprising administering to the mother of the fetus a polypeptidecomprising a chimeric polypeptide comprising a clotting factor portionand an Fc portion. In some embodiments, the clotting factor is FVIIzymogen, activated FVII, activatable FVII, or FIX.

In some embodiments, the administration of the chimeric polypeptidedecreases the incidence of spontaneous bleeding or prevents bleeding.

The present disclosure also provides a method of inducing immunetolerance to FVIII in a subject in need thereof, comprisingadministering to the subject a chimeric polypeptide comprising a FVIIIportion and an Fc portion. In some embodiments, the subject is at riskof developing an inhibitory FVIII immune response. In other embodiments,the subject has developed an inhibitory Factor VIII immune response.

Also provided is method of preventing or inhibiting development of aninhibitor to FVIII, the method comprising administering to a subject inneed of immune tolerance a chimeric polypeptide comprising FVIII portionand an Fc portion.

The present disclosure also provides a method of inducing immunetolerance to a clotting factor in a subject in need thereof, comprisingadministering to the subject a chimeric polypeptide comprising aclotting factor portion and an Fc portion. In some embodiments, thesubject is at risk of developing an inhibitory clotting factor immuneresponse. In other embodiments, the subject has developed an inhibitoryclotting factor immune response. In some embodiments, the clottingfactor portion comprises Factor VII, Factor IX or Von Willebrand factor.

Also provided is method of preventing or inhibiting development of aninhibitor to a clotting factor comprising administering to a subject inneed thereof a chimeric polypeptide comprising a clotting factor portionand an Fc portion. In some embodiments, the clotting factor portion isFactor VII, Factor IX or Von Willebrand factor.

In some embodiments, the subject is a human and the method ofadministering FVIII is a method for treating a bleeding condition insaid subject. In some embodiments, the bleeding condition is caused by ablood coagulation disorder. In some embodiments, the blood coagulationdisorder is hemophilia or von Willebrand disease. In some embodiments,the blood coagulation disorder is hemophilia A. In some embodiments, thesubject has a condition requiring prophylactic or on-demand treatment,such as a bleeding episode. In some embodiments, the subject is apatient who is suffering from a bleeding disorder or is expected to bein need of such treatment.

The present disclosure also provides a kit comprising: (a) apharmaceutical composition comprising a chimeric polypeptide whichcomprises a clotting factor portion and an Fc portion or an FcRn bindingpartner portion and a pharmaceutically acceptable carrier, and (b)instructions to administer to the composition to a subject in need ofimmune tolerance to the clotting factor. In some embodiments, thechimeric polypeptide comprises a FVIII portion, a FVII portion, or a FIXportion. In some embodiments, the chimeric polypeptide is a FVIIImonomer dimer hybrid, a FVII monomer dimer hybrid, or a FIX monomerdimer hybrid. In some embodiments, the instructions further include atleast one step to identify a subject in need of immune tolerance to theclotting factor. In some embodiments, the step to identify the subjectsin need of immune tolerance includes one or more from the groupconsisting of: (a) identifying a subject having a mutation or deletionin the clotting factor gene; (b) identifying a subject having arearrangement in the clotting factor gene; (c) identifying a subjecthaving a relative who has previously developed an inhibitory immuneresponse against the clotting factor; (d) identifying a subjectreceiving interferon therapy; (e) identifying a subject receivinganti-viral therapy; (f) identifying a subject having a genetic mutationin a gene other than the gene encoding the clotting factor which islinked with an increased risk of developing an inhibitory immuneresponse; and (g) two or more combinations thereof. In some embodiments,the genetic mutation in a gene other than the gene encoding the clottingfactor comprises one or more mutations selected from the groupconsisting of: (a) a genetic polymorphism associated with increasedTNF-α; (b) a genetic polymorphism associated with increased IL10; (c) agenetic polymorphism associated with decreased CTLA-4; (d) a mutation inDR15 or DQB0602 MHC Class II molecules; and (e) has two or morecombinations thereof.

In some embodiments, the methods of the present disclosure furthercomprise measuring the level of an inhibitory immune response after theadministration. In some embodiments, the methods of the presentdisclosure further comprise comparing the level of the inhibitory immuneresponse after the administration with the level of the inhibitoryimmune response before the administration. In some embodiments, theinhibitory immune response is development of antibodies against FVIII.In some embodiments, the inhibitory immune response is cytokinesecretion.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows a schematic representation of the rFVIIIFc monomer.

FIGS. 2A and 2B show non-reducing and reducing SDS-PAGE analysis ofrFVIIIFc (processed or single chain). FIG. 2C shows the rFVIIIFcstructure analyzed by LC/UV and LC/MS.

FIGS. 3A, 3B and 3C show the biochemical characterization of rFVIII-Fc.FIG. 3A shows the activation of Factor X (FX) as a function ofphospholipid vesicle concentration. FIG. 3B shows the activation of FXas a function of FX concentration. FIG. 3C shows the activation of FX asa function of activated FIX (FIXa) concentration.

FIG. 4 shows the activation of FX following cleavage by activatedProtein C.

FIGS. 5A, 5B, 5C and 5D show observed group mean FVIII activity (±SE)versus time profiles. Profiles are sorted by dose level, grouped bycompound versus time. FIG. 5A corresponds to a one stage assay with a 25IU/kg dose. FIG. 5B corresponds to a one stage assay with a 65 IU/kgdose. FIG. 5C corresponds to a chromogenic assay with a 25 IU/kg dose.FIG. 5D corresponds to a chromogenic assay with a 65 IU/kg dose.

FIGS. 6A and 6B show observed group mean FVIII activity (±SE) versustime profiles, grouped by dose level and compound versus time. FIG. 6Acorresponds to a one stage assay. FIG. 6B corresponds to a chromogenicassay.

FIGS. 7A, 7B and 7C show in vivo efficacy of single chain FVIIIFc (SCrFVIIIFc) in haemophilia A (HemA) mouse tail vein transection model.FIG. 7A shows the relationship between FVIII dose and protection. Singlechain rFVIIIFc doses are shown as squares, and processed rFVIIIFc dosesare shown as circles. FIG. 7B shows the percentage of survival followingtail vein transection after administration of 4.6 μg/kg, 1.38 μg/kg, and0.46 μg/kg of rFVIIIFc or SC rFVIIIFc. FIG. 7C shows the percentage ofnon-bleeders following tail vein transection, after administration of4.6 μg/kg (black circle or inverted triangle), 1.38 μg/kg (triangle ordiamond), and 0.46 μg/kg (square and gray circle) of rFVIIIFc or SCrFVIIIFc, respectively.

FIG. 8 depicts the study design of the phase 1/2a study, which was adose-escalation, sequential design to evaluate the safety and PK ofrFVIIIFc compared with ADVATE® after a single intravenous dose of either25 IU/kg (low dose Cohort A) or 65 IU/kg (high dose Cohort B).

FIG. 9 shows the correlation of rFVIII Activity by one-stage (aPTT) andchromogenic assays. Results measure FVIII activity (IU/mL) followinginjection of ADVATE® (♦) and rFVIIIFc (□).

FIGS. 10A and 10B show group mean plasma FVIII activity pharmacokineticprofiles for low-dose and high-dose cohorts. The plasma FVIII activities(one stage aPTT assay) versus time curve after a single intravenousinjection of rFVIIIFc or ADVATE® are shown for 25 IU/kg (low-dosecohort, n=6) (FIG. 10A); and 65 IU/kg (high dose cohort, n=10 [ADVATE®];n=9 [rFVIIIFc]) (FIG. 10B). Results presented are group mean±standarderror of mean (SEM).

FIGS. 11A and 11B show the effect of VWF antigen levels on Cl andt_(1/2) of FVIII activity after Injection of ADVATE® or rFVIIIFc. Thecorrelation between VWF antigen levels and the weight-adjusted Cl ofADVATE® (R²=0.5415 and p=0.0012) and rFVIIIFc (R²=0.5492 and p=0.0016)(FIG. 11A); and the t_(1/2) of ADVATE® (R²=0.7923 and p<0.0001) andrFVIIIFc (R²=0.6403 and p=0.0003) (FIG. 11B) are shown. Each dotrepresents an individual subject.

FIGS. 12A and 12 B show ex vivo whole blood ROTEM® results forindividual subjects after injection of ADVATE® or rFVIIIFc. Blood wassampled from subjects prior to and after treatment at doses of 25 IU/kgADVATE® and rFVIIIFc (FIG. 12A); and 65 IU/kg ADVATE® and rFVIIIFc atspecified time points (FIG. 12B). Clotting time was determined by NATEMinitiated with Ca on a ROTEM® instrument. Results presented aremean±standard error of mean (SEM) from triplicate channel readings foreach individual sample.

FIGS. 13A and 13B compare the activity of rFVIIIFc and SC rFVIIIFc in athrombin generation assay (TGA). FIG. 13A compared endogenous thrombinpotential (ETP) for rFVIIIFc, SC rFVIIIFc, fully processed rFVIIIFc andWHO standard FVIII. FIG. 13B compares peak thrombin for rFVIIIFc, SCrFVIIIFc, fully processed rFVIIIFc and WHO standard FVIII.

FIG. 14 shows a schematic representation of rFVIIIFc monomer. rFVIIIFcis a recombinant fusion of human B-domain deleted FVIII with Fc fromhuman IgG1, with no intervening linker sequence.

FIGS. 15A, 15B, 15C and 15D show pharmacokinetic (PK) profiles comparingrFVIIIFc and rFVIII in HemA mice (FIG. 15A), C57BL/6 mice (FIG. 15B),FcRn KO mice (FIG. 15C), and human FcRn transgenic Tg32B mice (FIG. 15D)following a tail vein injection of 125 IU/kg. Results shown are Mean±SDfrom 4 mice per treatment at each time point. The PK parameter estimatesare summarized in TABLE 12.

FIG. 16 compares the acute activity of rFVIIIFc and rFVIII in a HemAmice tail clip bleeding model. Male HemA mice received a tail veininjection of 24 IU/kg, 72 IU/kg, or 216 IU/kg of rFVIIIFc or rFVIIIfollowed by a 10 mm tail clip 5 minutes post dosing. Results presentedare individual and median blood loss over 30 minutes following the tailclip from 20 mice in each treatment group. P<0.05 for Vehicle vs. allother treatments, and P>0.05 for C57Bl/6 mice vs. HemA mice treated with72 or 216 IU/kg of rFVIIIFc, or 216 IU/kg of rFVIII.

FIGS. 17A and 17B shows the prophylactic efficacy of rFVIIIFc relativeto rFVIII in the tail vein transection (TVT) bleeding model. Male HemAmice were injured by TVT either 24 hours following vehicle, rFVIII, orrFVIIIFc treatment, or 48 hours following rFVIIIFc treatment. FIG. 17Ashows survival following TVT. P<0.001 by Log-Rank test of the survivalcurves from animals that received 12 IU/kg rFVIIIFc vs. rFVIII 24 hoursprior to TVT. FIG. 17B shows rebleed within 24 hours following TVT.P=0.002 by Log-Rank test of the non-rebleed curves from animals thatreceived 12 IU/kg rFVIIIFc vs. rFVIII 24 hours prior to TVT.

FIGS. 18A and 18B show whole blood clotting time (WBCT) of rFVIIIFc andrFVIII in hemophilia A dogs. Normal WBCT range in dogs is shown by thelarge dashed lines. The area above the small dashed lines (20 minutes)indicates the point at which the plasma FVIII activity is expected to bebelow 1% of normal. FIG. 18A shows WBCT after administration ofrFVIIIFc. FIG. 18B shows WBCT after administration of rFVIII followed byadministration of rFVIIIFc in a crossover study.

FIGS. 19A and 19B present pharmacokinetics (PK) data for rFVIIIFccompared to rFVIII in Hemophilia A dogs after an i.v. dose. FIG. 19Ashows plasma antigen concentration measured by ELISA. FIG. 19B showsplasma FVIII activity was measured by chromogenic assay. N=4 forrFVIIIFc and N=2 for rFVIII.

FIG. 20 shows the design of a study to evaluate FVIII Immunogenicity inHemA mice. Intravenous (i.v.) dosing with rFVIIIFc; chimeric humanFVIII-murine Fc; BDD-FVIII (XYNTHA®); full-length rFVIII (ADVATE®); andvehicle control took place at day 0, day 7, day 14, day 21 and day 35.Blood samples were collected a day 14, day 21, day 28, day 35 and day42.

FIGS. 21A, 21B and 21C show anti-FVIII total antibody counts inimmunogenicity experiments conducted in HemA mice according to thedesign study shown in FIG. 20. FIG. 21A corresponds to total anti-FVIIIantibody measurements after repeated i.v. dosing with 50 IU/kg rFVIIIFc;chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA®); or full-lengthrFVIII (ADVATE®). FIG. 21B corresponds to total anti-FVIII antibodymeasurements after repeated i.v. dosing with 100 IU/kg rFVIIIFc;chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA®); full-length rFVIII(ADVATE®). FIG. 21C corresponds to total anti-FVIII antibodymeasurements after repeated i.v. dosing with 250 IU/kg rFVIIIFc;chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA®); full-length rFVIII(ADVATE®).

FIGS. 22A, 22B, 22C, and 22D shows anti-FVIII antibody measurements atdifferent times after administration of 50 IU/kg, 100 IU/kg, and 250IU/kg doses of rFVIIIFc; chimeric human FVIII-murine Fc; BDD-FVIII(XYNTHA®); full-length rFVIII (ADVATE®). FIG. 22A shows datacorresponding to samples collected at day 14. FIG. 22B shows datacorresponding to samples collected on day 21. FIG. 22C shows datacorresponding to samples collected on day 28. FIG. 22D shows datacorresponding to samples collected on day 42.

FIG. 23 shows the correlation between total and neutralizing antibodiesto FVIII.

FIG. 24 shows anti-hFc antibody development after treatment with 50IU/kg and 250 IU/kg doses of rFVIIIFc (rFVIII with human Fc).

FIG. 25 is a diagram showing the experimental procedures for theisolation and analysis of splenocytes from HemA mice in a study tomeasure the splenic lymphocyte response to rFVIIIFc compared withcommercially available FVIII.

FIG. 26 is a diagram showing the procedure for intracellular cytokinestaining using FACS (fluorescence-activated cell sorting). The procedureuses five colors, one for the CD4 lymphocyte marker, and another fourcolors for the IL2, IL-4, IL-10, and TNF-α cytokines.

FIGS. 27A and 27B show representative FACS dot plot. FIG. 27Acorresponds to the intracellular cytokine staining of the isotypecontrol. FIG. 27B corresponds to the intracellular cytokine staining ofdouble positive cell containing the CD4 and TNF-α markers.

FIG. 28 shows intracellular cytokine staining above control (vehicle)for CD4 and interleukin-2 (IL-2). Percentages of double positive cellswere determined from FACS dot plots from all the FVIII treatments andvehicle. Percent of double positive cells in FVIII treated mice wasobtained by comparing with vehicle treated group. The samples correspondto rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA® (Xyn), andADVATE® (Adv). FVIII treatments were administered at 50 IU/kg and 250IU/kg doses.

FIG. 29 shows intracellular cytokine staining above control (vehicle)for CD4 and TNF-α. Percentages of double positive cells were determinedfrom FACS dot plots from all the FVIII treatments and vehicle. Percentof double positive cells in FVIII treated mice was obtained by comparingwith vehicle treated group. The samples correspond to rFVIIIFc with ahuman Fc (hFc), or mouse Fc (mFc), XYNTHA® (Xyn), and ADVATE® (Adv).FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

FIG. 30 shows intracellular cytokine staining above control (vehicle)for CD4 and interleukin-4 (IL-4). Percentages of double positive cellswere determined from FACS dot plots from all the FVIII treatments andvehicle. Percent of double positive cells in FVIII treated mice wasobtained by comparing with vehicle treated group. The samples correspondto rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA® (Xyn), andADVATE® (Adv). FVIII treatments were administered at 50 IU/kg and 250IU/kg doses.

FIG. 31 shows intracellular cytokine staining above control (vehicle)for CD4 and interleukin-10 (IL-10). Percentages of double positive cellswere determined from FACS dot plots from all the FVIII treatments andvehicle. Percent of double positive cells in FVIII treated mice wasobtained by comparing with vehicle treated group. The samples correspondto rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA® (Xyn), andADVATE® (Adv). FVIII treatments were administered at 50 IU/kg and 250IU/kg doses.

FIG. 32 shows intracellular cytokine staining above control (vehicle)for CD4 and dendritic cell marker PD-L1 (CD274). Percentages of doublepositive cells were determined from FACS dot plots from all the FVIIItreatments and vehicle. Percent of double positive cells in FVIIItreated mice was obtained by comparing with vehicle treated group. Thesamples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc),XYNTHA® (Xyn), and ADVATE® (Adv). FVIII treatments were administered at50 IU/kg and 250 IU/kg doses.

FIG. 33 shows intracellular cytokine staining above control (vehicle)for CD4 and dendritic cell marker CD80. Percentages of double positivecells were determined from FACS dot plots from all the FVIII treatmentsand vehicle. Percent of double positive cells in FVIII treated mice wasobtained by comparing with vehicle treated group. The samples correspondto rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA® (Xyn), andADVATE® (Adv). FVIII treatments were administered at 50 IU/kg and 250IU/kg doses.

FIG. 34 shows intracellular cytokine staining above control (vehicle)for CD4 and Treg marker Foxp3. Percentages of double positive cells weredetermined from FACS dot plots from all the FVIII treatments andvehicle. Percent of double positive cells in FVIII treated mice wasobtained by comparing with vehicle treated group. The samples correspondto rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA® (Xyn), andADVATE® (Adv). FVIII treatments were administered at 50 IU/kg and 250IU/kg doses.

FIG. 35 shows intracellular cytokine staining above control (vehicle)for CD4 and the Th inhibitory molecule Tim3. Percentages of doublepositive cells were determined from FACS dot plots from all the FVIIItreatments and vehicle. Percent of double positive cells in FVIIItreated mice was obtained by comparing with vehicle treated group. Thesamples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc),XYNTHA® (Xyn), and ADVATE® (Adv). FVIII treatments were administered at50 IU/kg and 250 IU/kg doses.

FIG. 36 shows intracellular cytokine staining above control (vehicle)for CD4 and the Th inhibitory molecule CD279 (PD-1). Percentages ofdouble positive cells were determined from FACS dot plots from all theFVIII treatments and vehicle. Percent of double positive cells in FVIIItreated mice was obtained by comparing with vehicle treated group. Thesamples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc),XYNTHA® (Xyn), and ADVATE® (Adv). FVIII treatments were administered at50 IU/kg and 250 IU/kg doses.

FIG. 37 is a diagram showing the design of the immunogenicity studypresented in Example 13. FVIII (rFVIIIFc, XYNTHA®, ADVATE®) and vehiclecontrol were administered to HemA mice on day 0, day 7, day 14, day 21,day 35 and day 53. Blood was drawn on day 0, day 14, day 21, day 28, andday 42. Spleens were collected on day 56. FVIII was administered in 50IU/kg, 100 IU/kg, and 250 IU/kg doses.

FIG. 38 shows the methodology used for analysis of mouse splenocytes andT-cell response profiling in Example 13.

FIG. 39 shows total anti-FVIII antibody levels in blood samplescollected on day 42. rFVIIIFc, BDD-rFVIII (XYNTHA®) and fl-rFVIII(ADVATE®) were administered in 50 IU/kg, 100 IU/kg, and 250 IU/kg doses.

FIGS. 40A, 40B and 40C show intracellular cytokine staining of splenicCD4+ T-cells from HemA mice treated with different doses of FVIII(rFVIIIFc, BDD-rFVIII (XYNTHA®) or fl-rFVIII (ADVATE®)). Each figureshows results for IL-2 (left panel) and for TNF-α (right panel). FIG. 4Acorresponds to HemA mice (N=4) injected with 50 IU/kg doses of FVIII.FIG. 4B corresponds to HemA mice (N=10) injected with 100 IU/kg doses ofFVIII. FIG. 4C corresponds to HemA mice (N=4) injected with 250 IU/kgdoses of FVIII.

FIG. 41 shows intracellular cytokine staining for CD4/CD25/Foxp3 triplepositive splenocytes isolated from HemA mice treated with 100 IU/kgdoses of rFVIIIFc, BDD-rFVIII (XYNTHA®) or fl-rFVIII (ADVATE®).

FIGS. 42A-H show real time PCR for immune tolerance related cytokines in100 IU/kg-treated HemA mice. FIG. 42A shows results for TGF-β, FIG. 42Bshows results for interleukin-10, FIG. 42C shows results for the IL-12asubunit of IL-35, and FIG. 42D shows results for the EBI-3 subunit ofIL-35. FIG. 42E shows results for Foxp3. FIG. 42F shows results forIL2ra/CD25. FIG. 42G shows results for CTLA4. FIG. 42H shows results forIDO-1.

FIG. 43 shows FACS analysis of cells involved in the PD-L1-PD-1 pathwayin 100 IU/kg treated mice. Splenocytes were stained for either surfaceCD11c and PD-L1 (FIG. 43A), or CD4 and PD-1 (FIG. 43B). Bars representpercent over vehicle (*p<0.05 vs. vehicle; +p<0.05 between treatments;T-test).

FIG. 44 is a diagram showing the design of an immunogenicity comparisonstudy for rFVIIIFc, XYNTHA® and ADVATE® in HemA Mice. FVIII doses wereadministered to HemA mice on day 0, day 7, day 14, day 21, and day 35.Blood was drawn on day 0, day 14, day 21, day 28, and day 42. FVIII wasadministered in 50 IU/kg, 100 IU/kg, and 250 IU/kg doses.

FIG. 45 shows anti-FVIII antibody measurements at 14, 21, 28 and 42 daysafter administration of 50 IU/kg doses of rFVIIIFc; BDD-FVIII (XYNTHA®);full-length rFVIII (ADVATE®), as well as levels of inhibitory antibodiesat day 42.

FIG. 46. shows anti-FVIII antibody measurements at 14, 21, 28 and 42days after administration of 100 IU/kg doses of rFVIIIFc; BDD-FVIII(XYNTHA®); full-length rFVIII (ADVATE®), as well as levels of inhibitoryantibodies at day 42.

FIG. 47 shows anti-FVIII antibody measurements at 14, 21, 28 and 42 daysafter administration of 250 IU/kg doses of rFVIIIFc; BDD-FVIII(XYNTHA®); full-length rFVIII (ADVATE®), as well as levels of inhibitoryantibodies at day 42.

FIG. 48 shows the correlation between FVIII neutralizing antibody titersand total binding antibody levels after administration of rFVIII andrFVIIIFc.

FIG. 49 is a diagram showing the T-cell response profiling component ofthe immunogenicity comparison study for rFVIIIFc, XYNTHA® and ADVATE® inHemA mice shown in FIG. 44. Additional FVIII doses were administered toHemA mice on day 53, and spleen were collected on day 56.

FIG. 50 (right panel) shows intracellular cytokine staining forCD4/CD25/Foxp3 triple positive splenocytes isolated from HemA micetreated with 100 IU/kg doses of rFVIIIFc, BDD-rFVIII (XYNTHA®) orfl-rFVIII (ADVATE®). FIG. 50 (left panel) is a diagram showing themechanism of action of regulatory T-cells.

FIG. 51 is a diagram showing the design of an rFVIIIFc immunetolerization study. 50 IU/kg doses of rFVIIIFc were administered i.v. toHemA mice on day 0, day 7, day, day 21, and day 35. Blood was drawn onday 0, day 14, day 21, day 28, and day 42, followed by a 1 week restperiod. Mice were then rechallenged with 250 IU/kg doses of rFVIIIFc onday 49 (day 0 of rechallenge), day 56 (day 7 of rechallenge), day 63(day 14 of rechallenge), and day 70 (day 21 of rechallenge). Blood wascollected during the rechallenge on day 63 (day 14 of rechallenge), day70 (day 21 of rechallenge), and day 77 (day 28 of rechallenge).

FIG. 52 shows total anti-FVIII antibody measurements at day 14, 21, and28 post rechallenge with 250 IU/kg doses of rFVIIIFc. HemA mice werepretreated with 50 IU/kg rFVIIIFc or vehicle control. The resultsindicate that rFVIIIFc induces immune tolerance in HemA mice.

FIG. 53 is a diagram showing the recycling of IgG and rFVIIIFc by FcRn.

FIG. 54 is a diagram depicting cell types and cellular architecturesurrounding a liver sinusoid.

FIG. 55 is a diagram showing the design of a clearance assay in whichmacrophage and Kupffer are depleted with CLODROSOME® (ENCAPSOME®administered as control) prior to i.v. injection of FVIII or rFVIIIFc.Three knock-out mouse models were used: HemA, DKO, and FcRn-KO. Bloodsamples were collected at the specified time point (4 samples per timepoint).

FIG. 56 shows a representative staining of HemA mouse liver section withan antibody to Iba-1, a specific macrophage marker. Panels A and A′ showcontrol ENCAPSOME® treatment of HemA mice. Panels B and B′ showCLODROSOME® treatment of HemA mice. Panels A′ and B′ show thequantification masks highlighting the stained Kupffer cells, totaltissue area, and empty areas.

FIG. 57 shows an immunohistochemical (IHC) quantitative analysis ofareas positively stained with a labeled antibody to F4/80 aftertreatment with CLODROSOME® or ENCAPSOME®, and a fluorescence-activatedcell sorter (FACS) analysis identifying circulating monocytic cells inblood cells stained with the same labeled antibody to F4/80.

FIG. 58 shows RT-PCR analysis of the expression of the macrophage markerepidermal growth factor module-containing mucin-like receptor 1 (Emr1)(F4/80) in the liver, spleen, and lung of HemA mice treated withENCAPSOME® or CLODROSOME®.

FIG. 59 shows clearance of rFVIII and rFVIIIFc in control HemA mice andmacrophage/Kupffer cell depleted HemA mice.

FIG. 60 shows clearance of rFVIII and rFVIIIFc in control DKO mice (micelacking FVIII and VWF) and macrophage/Kupffer cell depleted DKO mice.

FIG. 61 shows clearance of rFVIII and rFVIIIFc in control FcRn-KO mice(mice lacking the FcRn recycling receptor) and macrophage/Kupffer celldepleted FcRn-KO mice

FIG. 62 shows Bethesda titers of mice born out of mothers immunized ongestation day 16 with the indicated FVIII drug substance or Control(untreated). Panel A shows the experimental design, depicting thetimings of treatment and dose of rFVIIIFc or XYNTHA® (BDD-FVIII) topregnant mice and pups born out of them. Panels B and C show Bethesdatiters for rFVIIIFc from pups born out of rFVIIIFc-treated,XYNTHA®-treated, or Control (untreated) mice, grouped according totreatment cohort (Panel B) or grouped by individual mothers (Panel C).

FIG. 63 shows Bethesda titers of mice born out of mothers immunized ongestation day 15-17 with the indicated FVIII drug substance or control(untreated). Panel A shows the experimental design, depicting thetimings of treatment and dose of rFVIIIFc or XYNTHA® (BDD-FVIII) topregnant mice and pups born out of them. Panels B and C show Bethesdatiters for rFVIIIFc from pups born out of rFVIIIFc-treated,XYNTHA®-treated, or Control (untreated) mice, grouped according totreatment cohort (Panel B) or grouped by individual mothers (Panel C).

FIGS. 64A and 64B show dendritic cell surface expression of CD80 andCD274, respectively, determined by staining splenocytes from micetreated with 100 IU/kg rFVIIIFc, B-domain deleted FVIII (BDD-FVIII;XYNTHA®) or full length FVIII (fl-FVIII; ADVATE®) for these two antigensalong with CD11c and MHC Class II. The results show percentsplenocytes±SEM (n=7-9; *p<0.05 vs. vehicle; \p<0.05 vs. rFVIIIFc;T-test). FIGS. 64C and 64D show mRNA expression levels of CD274 andIDO1, respectively, determined by real time PCR from splenocytesnormalized to GAPDH levels. Bars represent relative expression levels(2^(−ΔCt))±SEM (n=4-9; *p<0.05 vs. vehicle; \p<0.05 vs. rFVIIIFc;T-test).

FIG. 65 shows a heat map depicting the expression profiles of all thegenes in a real time PCR array among the three tested groups, i.e.,splenocytes of vehicle, 50 IU/kg and 250 IU/kg rFVIIIFc treated HemAmice. cDNA from each of the samples was used to monitor the expressionof individual genes using a real time PCR array consisting of genesfocused on tolerance and anergy associated molecules.

FIG. 66 shows an expression profile of candidate genes that wereidentified as being up- or down-regulated by the splenocytes of 50 IU/kgrFVIIIFc treated HemA mice and comparison with the 250 IU/kg rFVIIIFctreated group. Results illustrate the change in expression of genesabove the vehicle group. The cut-off for fold change in regulation wastaken as 2, i.e., fold change above 2 was considered upregulation andbelow 0.5 as downregulation. All the candidate genes belonging to the 50IU/kg group shown here were significantly regulated (p<0.05 vs. vehicleas well as the 250 IU/kg group; n=8-11).

FIG. 67 shows a T-cell proliferation profile comparison between HemAmice receiving two weekly injections of either 50 IU/kg or 250 IU/kg ofrFVIIIFc. Bars represent decrease in MFI of CFSE relative to control inT-cells±SEM (*p<0.05, T-test, n=3-5) from the 250 IU/kg and 50 IU/kggroups.

FIG. 68A-D shows IFNγ secretion profiles of T-cells from HemA micereceiving two weekly injections of either 250 IU/kg of rFVIIIFc (FIG.68A), 50 IU/kg of rFVIIIFc (FIG. 68B), 250 IU/kg of rFVIIIFc-N297A (FIG.68C), or 50 IU/kg rFVIIIFc-N297A (FIG. 68D). Bars represent fold abovevehicle of IFNγ secretion±SEM (*p<0.05, T-test; n=3-5).

DETAILED DESCRIPTION

The present disclosure provides a method of treating Hemophilia A withFactor VIII (FVIII) (processed, single chain, or a combination thereof)using a longer dosing interval and/or greater AUC than is possible withcurrently known FVIII products. The present disclosure also providesmethods of inducing immune tolerance to FVIII. The present disclosurealso provides improved FVIII chimeric polypeptides and methods ofproduction.

The methods of inducing immune tolerance and production of improvedchimeric polypeptides disclosed herein are also generally applicable toone or more clotting factors, e.g., FVII and FIX. Accordingly, thepresent disclosures regarding FVIII chimeric polypeptides (e.g.,FVIIIFc) and their uses, are equally applicable to other chimericpolypeptides comprising a clotting factor portion and an Fc portion. Insome specific examples, the clotting factor portion of the chimericpolypeptide is FVII or FIX. In this respect, the present disclosureprovides in general a method of inducing immune tolerance to a clottingfactor in a subject in need thereof comprising administering to thesubject a chimeric polypeptide comprises a clotting factor portion andan Fc portion. Also provided is a method of preventing or inhibitingdevelopment of an inhibitor to a clotting factor comprisingadministering to a subject in need of immune tolerance to the clottingfactor a chimeric polypeptide, wherein the chimeric polypeptidecomprises a clotting factor portion and an Fc portion.

Treatment of hemophilia A is by replacement therapy targetingrestoration of FVIII activity to 1 to 5% of normal levels to preventspontaneous bleeding (Mannucci, P. M. et al., N. Engl. J. Med.344:1773-9 (2001), herein incorporated by reference in its entirety).There are plasma-derived and recombinant FVIII products available totreat bleeding episodes on-demand or to prevent bleeding episodes fromoccurring by treating prophylactically. Based on the short half-life ofthese products (8-12 hours) (White G. C., et al., Thromb. Haemost.77:660-7 (1997); Morfini, M., Haemophilia 9 (suppl 1):94-99; discussion100 (2003)), treatment regimens require frequent intravenousadministration, commonly two to three times weekly for prophylaxis andone to three times daily for on-demand treatment (Manco-Johnson, M. J.,et al., N. Engl. J. Med. 357:535-544 (2007)), each of which isincorporated herein by reference in its entirety. Such frequentadministration is painful and inconvenient. Another major challengeassociated with currently available FVIII products is the development ofneutralizing anti-FVIII antibodies in patients receiving FVIIItherapies. Inhibitory FVIII immune responses can comprise anti-FactorVIII antibodies and/or cell-mediated immune responses.

The present disclosure provides a method of administering FVIII to ahuman subject in need thereof (e.g., human patient) that is at risk ofdeveloping an inhibitory FVIII immune response. The method comprisesadministration of a chimeric polypeptide comprising a FVIII portion andan Fc portion.

In some embodiments, the administration of the chimeric polypeptidereduces the number of antibodies to FVIII in the subject compared to thenumber prior to administration. In some embodiments, the chimericpolypeptide is administered to a subject with an inhibitory FVIII immuneresponse, and the administration can reduce the inhibitory immuneresponse. The inhibitory FVIII immune response can be an inhibitoryantibody immune response and/or a cell-mediated immune response.Administration of a chimeric polypeptide according to the methodsprovided herein can decrease or neutralize the inhibitory immuneresponse. Thus, in some embodiments, anti-FVIII antibodies are decreasedor eliminated in a subject after administration of a chimericpolypeptide comprising a FVIII portion and an Fc portion. The decreasecan be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%reduction.

In some embodiments, the administration of the chimeric polypeptidereduces the titer of antibodies to FVIII in the subject compared to thetiter prior to administration. In some embodiments, the titer ofanti-FVIII antibodies is decreased in a subject after administration ofa chimeric polypeptide comprising a FVIII portion and an Fc portion.Accordingly, administration of a chimeric polypeptide comprising a FVIIIportion and an Fc portion can reduce inhibitors to less than 20, lessthan 10, less than 5, less than 4, less than 3, less than 2, less than1, or less than 0.6 Bethesda Units (BU).

In some embodiments, the administration of the chimeric polypeptidereduces the level of a cytokine in the subject compared to the levelprior to administration. In some embodiments, cytokine levels aredecreased in a subject after administration of a chimeric polypeptidecomprising a FVIII portion and an Fc portion. The cytokine can be, forexample, Il-12, IL-4, and/or TNF-α. The decrease can be, for example, a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction.

In some embodiments, the chimeric polypeptide is administered to asubject that previously developed an inhibitory FVIII immune response.Administration of a chimeric polypeptide to such subjects, according tothe methods provided herein, can result in a decreased immune responsecompared to the previous response. Thus, in some embodiments, feweranti-FVIII antibodies are produced in a subject after administration ofa chimeric polypeptide according to the present methods than wereproduced after administration of a polypeptide consisting of a FVIIIpolypeptide. In some embodiments, the administration of the chimericpolypeptide reduces the number of antibodies to FVIII in the subjectcompared to the number in the subject after a previous treatment with apolypeptide consisting of a FVIII polypeptide.

In some embodiments, the administration of the chimeric polypeptidereduces the titer of antibodies to FVIII in the subject compared to thetiter in the subject after a previous treatment with a polypeptideconsisting of a FVIII polypeptide. In some embodiments, the titer ofanti-FVIII antibodies is lower in a subject after administration of achimeric polypeptide comprising a FVIII portion and an Fc portion thanwas produced after administration of a polypeptide consisting of a FVIIIpolypeptide. In some embodiments, the administration of the chimericpolypeptide reduces the level of a cytokine in the subject compared tothe level in the subject after a previous treatment with a polypeptideconsisting of a FVIII polypeptide. In some embodiments, cytokine levels(e.g., IL-12, IL-4, and/or TNF-α) are lower in a subject afteradministration of a chimeric polypeptide comprising a FVIII portion andan Fc portion than the levels after administration of a polypeptideconsisting of a FVIII polypeptide.

In some embodiments, the administration of the chimeric polypeptidereduces the number of anti-clotting factor antibodies in the subjectcompared to the number that would result from administration of apolypeptide consisting of the clotting factor portion or a polypeptidecomprising the clotting factor portion, but not comprising the Fcportion to the subject. In some embodiments, the chimeric polypeptide isadministered to a subject that has not previously developed aninhibitory FVIII immune response. Administration of a chimericpolypeptide to such subjects, according to the methods provided herein,can result in a lower immune response than would result fromadministration of a polypeptide consisting of a FVIII polypeptide. Thus,in some embodiments, fewer anti-FVIII antibodies are produced in asubject after administration of a chimeric polypeptide according to thepresent methods than would be produced by administration of apolypeptide consisting of a FVIII polypeptide.

In some embodiments, the administration of the chimeric polypeptidereduces the titer of anti-clotting factor antibodies in the subjectcompared to the titer that would result from administration of apolypeptide consisting of the clotting factor portion or a polypeptidecomprising the clotting factor portion, but not comprising the Fcportion to the subject. In some embodiments, the titer of anti-FVIIIantibodies is lower in a subject after administration of a chimericpolypeptide comprising a FVIII portion and an Fc portion than would beproduced by administration of a polypeptide consisting of a FVIIIpolypeptide. In some embodiments, the administration of the chimericpolypeptide reduces the level of a cytokine (e.g., IL-12, IL-4, and/orTNF-α) in the subject compared to the level that would result fromadministration of a polypeptide consisting of the clotting factorportion or a polypeptide comprising the clotting factor portion, but notcomprising the Fc portion to the subject. In some embodiments, cytokinelevels (e.g., IL-12, IL-4, and/or TNF-α) are lower in a subject afteradministration of a chimeric polypeptide comprising a FVIII portion andan Fc portion than they would be after administration of a polypeptideconsisting of a FVIII polypeptide.

The methods of the present disclosure can further comprise, prior toadministration of the chimeric polypeptide, identifying that the subjecthas one or more characteristics selected from the group consisting of:(a) has a mutation or deletion in the gene encoding the clotting factor;(b) has a rearrangement in the gene encoding the clotting factor; (c)has a relative who has previously developed an inhibitory immuneresponse against the clotting factor; (d) is receiving interferontherapy; (e) is receiving anti-viral therapy; (f) has a genetic mutationin a gene other than the gene encoding the clotting factor which islinked with an increased risk of developing an inhibitory immuneresponse; and, (g) has two or more combinations thereof. In someembodiments, the subject has a genetic mutation in a gene other than thegene encoding the clotting factor comprises one or more mutationselected from the group consisting of: (i) a genetic polymorphismassociated with increased TNF-α; (ii) a genetic polymorphism associatedwith increased IL10; (iii) a genetic polymorphism associated withdecreased CTLA-4; (iv) a mutation in DR15 or DQB0602 MHC Class IImolecules; and, (v) two or more combinations thereof. In someembodiments, the polymorphism is associated with increased TNF-α is308G>A. In some embodiments, the polymorphism associated with increasedIL10 is allele 134 of the IL10G microsatellite.

The present disclosure also provides a method of administering FVIII toa human subject in need thereof (e.g., human patient), comprisingadministering to the subject a therapeutic dose of a chimeric FVIIIpolypeptide, e.g., a chimeric FVIII-Fc polypeptide, or a hybrid of sucha polypeptide at a dosing interval at least about one and one-half timeslonger than the dosing interval required for an equivalent dose of saidFVIII without the non-FVIII portion (a polypeptide consisting of saidFVIII portion), e.g., without the Fc portion. The present disclosure isalso directed to a method of increasing dosing interval of FVIIIadministration in a human subject in need thereof comprisingadministering the chimeric FVIII polypeptide.

The dosing interval can be at least about one and one-half to six timeslonger, one and one-half to five times longer, one and one-half to fourtimes longer, one and one-half to three times longer, or one andone-half to two times longer, than the dosing interval required for anequivalent dose of said FVIII without the non-FVIII portion (apolypeptide consisting of said FVIII portion), e.g., without the Fcportion. The dosing interval can be at least about one and one-half,two, two and one-half, three, three and one-half, four, four andone-half, five, five and one-half or six times longer than the dosinginterval required for an equivalent dose of said FVIII without thenon-FVIII portion (a polypeptide consisting of said FVIII portion),e.g., without the Fc portion. The dosing interval can be about everythree, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, or fourteen days or longer.

The dosing interval can be at least about one and one-half to 5, one andone-half, 2, 3, 4, or 5 days or longer.

The present disclosure also provides a method of administering FVIII toa human subject in need thereof, comprising administering to the subjecta therapeutic dose of a chimeric FVIII polypeptide, e.g., a chimericFVIII-Fc polypeptide, or a hybrid of such a polypeptide to obtain anarea under the plasma concentration versus time curve (AUC) at leastabout one and one-quarter times greater than the AUC obtained by anequivalent dose of said FVIII without non-FVIII portion (a polypeptideconsisting of said FVIII portion), e.g., without the Fc portion. Thepresent disclosure thus includes a method of increasing or extending AUCof FVIII activity in a human patient in need thereof comprisingadministering the chimeric FVIII polypeptide.

The present disclosure also provides a method of administering FVIII toa subject in need thereof, comprising administering to the subject atherapeutic dose of a polypeptide comprising a FVIII and an Fc or ahybrid of such a polypeptide at a dosing interval of about every three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, orfourteen days or longer.

The methods disclosed herein can be practiced on a subject in need ofprophylactic treatment or on-demand treatment.

“Administering,” as used herein, means to give a pharmaceuticallyacceptable FVIII polypeptide disclosed herein to a subject via apharmaceutically acceptable route. Routes of administration can beintravenous, e.g., intravenous injection and intravenous infusion.Additional routes of administration include, e.g., subcutaneous,intramuscular, oral, nasal, and pulmonary administration. Chimericpolypeptides and hybrid proteins can be administered as part of apharmaceutical composition comprising at least one excipient.

“Area under the plasma concentration versus time curve (AUC),” as usedherein, is the same as the term of art in pharmacology, and is basedupon the rate and extent of absorption of FVIII followingadministration. AUC is determined over a specified time period, such as12, 18, 24, 36, 48, or 72 hours, or for infinity using extrapolationbased on the slope of the curve. Unless otherwise specified herein, AUCis determined for infinity. The determination of AUC can be carried outin a single subject, or in a population of subjects for which theaverage is calculated.

A “B domain” of FVIII, as used herein, is the same as the B domain knownin the art that is defined by internal amino acid sequence identity andsites of proteolytic cleavage by thrombin, e.g., residues Ser741-Arg1648of full length human FVIII. The other human FVIII domains are defined bythe following amino acid residues: A1, residues Ala1-Arg372; A2,residues Ser373-Arg740; A3, residues Ser1690-Ile2032; C1, residuesArg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequenceincludes residues Ser1690-Tyr2332. The remaining sequence, residuesGlu1649-Arg1689, is usually referred to as the FVIII light chainactivation peptide. The locations of the boundaries for all of thedomains, including the B domains, for porcine, mouse and canine FVIIIare also known in the art. In one embodiment, the B domain of FVIII isdeleted (“B domain deleted FVIII” or “BDD FVIII”).

An example of a BDD FVIII is REFACTO® (recombinant BDD FVIII), which hasthe same sequence as the FVIII portion of the sequence in TABLE 2A(i)(amino acids 1 to 1457 or 20 to 1457 of SEQ ID NO:2). In anotherembodiment, the B domain deleted FVIII contains an intact intracellularprocessing site, which corresponds to Arginine at residue 754 of Bdomain deleted FVIII, which corresponds to Arginine residue 773 of SEQID NO: 2, or residue 1648 of full-length FVIII, which corresponds toArginine residue 1667 of SEQ ID NO: 6. The sequence residue numbers usedherein without referring to any SEQ ID Numbers correspond to the FVIIIsequence without the signal peptide sequence (19 amino acids) unlessotherwise indicated. For example, S743/Q1638 of full-length FVIIIcorresponds to S762/Q1657 of SEQ ID NO: 6 due to the 19 amino acidsignal peptide sequence.

A “B domain deleted FVIII” can have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In someembodiments, a B domain deleted FVIII sequence comprises any one of thedeletions disclosed at col. 4, line 4 to col. 5, line 28 and examples1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No. 6,346,513). Insome embodiments, a B domain deleted FVIII has a deletion disclosed atcol. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (alsoU.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No.6,228,620).

In some embodiments, a B domain deleted FVIII has a deletion describedin col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col.6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 ofU.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 ofU.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3,line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col.11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4,lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments, a B domaindeleted FVIII has a deletion of most of the B domain, but still containsamino-terminal sequences of the B domain that are essential for in vivoproteolytic processing of the primary translation product into twopolypeptide chain (i.e., intracellular processing site), as disclosed inWO 91/09122, which is incorporated herein by reference in its entirety.

In some embodiments, a B domain deleted FVIII is constructed with adeletion of amino acids 747-1638, i.e., virtually a complete deletion ofthe B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323(1990), incorporated herein by reference in its entirety. A B domaindeleted FVIII can also contain a deletion of amino acids 771-1666 oramino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4):301-6 (1988), incorporated herein by reference in its entirety.Additional B domain deletions that are part of the instant disclosureinclude, e.g., deletion of amino acids 982 through 1562 or 760 through1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. 83:5939-5942 (1986)),797 through 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), 741through 1646 (Kaufman (PCT published application No. WO 87/04187)),747-1560 (Sarver et al., DNA 6:553-564 (1987)), 741 through 1648 (Pasek(PCT application No. 88/00831)), 816 through 1598 or 741 through 1689(Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each ofwhich is incorporated herein by reference in its entirety. Each of theforegoing deletions can be made in any FVIII sequence.

In one embodiment, the B domain deleted FVIII portion in the chimericpolypeptide is processed into two chains connected (or associated) by ametal bond, the first chain comprising a heavy chain (A1-A2-partial B)and a second chain comprising a light chain (A3-C1-C2). In anotherembodiment, the B domain deleted FVIII portion is a single chain FVIII.The single chain FVIII can comprise an intracellular processing site,which corresponds to Arginine at residue 754 of B domain deleted FVIII(residue 773 of SEQ ID NO: 2) or at residue 1648 of full-length FVIII(residue 1657 of SEQ ID NO: 6).

The metal bond between the heavy chain and the light chain can be anymetal known in the art. For example, the metal can be a divalent metalion. The metals that can be used to associate the heavy chain and lightchain include, but not limited to, Ca²⁺, Mn²⁺, or Cu²⁺. Fatouros et al.,Intern. J. Pharm. 155(1): 121-131 (1997); Wakabayashi et al., JBC.279(13): 12677-12684 (2004).

In some embodiments, the FVIII portion in the chimeric polypeptidecomprises the A1 domain of FVIII. In some embodiments, the FVIII portionin the chimeric polypeptide comprises the A2 domain of FVIII. In someembodiments, the FVIII portion in the chimeric polypeptide comprises theA3 domain of FVIII. In some embodiments, the FVIII portion in thechimeric polypeptide comprises the C1 domain of FVIII. In someembodiments, the FVIII portion in the chimeric polypeptide comprises theC2 domain of FVIII.

“Chimeric polypeptide,” as used herein, means a polypeptide thatincludes within it at least two polypeptides (or subsequences orpeptides) from different sources. Chimeric polypeptides can include,e.g., two, three, four, five, six, seven, or more polypeptides fromdifferent sources, such as different genes, different cDNAs, ordifferent animal or other species. Chimeric polypeptides can include,e.g., one or more linkers joining the different subsequences. Thus, thesubsequences can be joined directly or they can be joined indirectly,via linkers, or both, within a single chimeric polypeptide. Chimericpolypeptides can include, e.g., additional peptides such as signalsequences and sequences such as 6His and FLAG that aid in proteinpurification or detection. In addition, chimeric polypeptides can haveamino acid or peptide additions to the N- and/or C-termini.

In some embodiments, the chimeric polypeptide comprises a clottingfactor portion (e.g., a FVIII portion) and a non-clotting factor portion(e.g., a non-FVIII portion). Exemplary non-clotting factor portions(e.g., non-FVIII portions) include, for example, Fc. Exemplary chimericFVIII-Fc polypeptides include, e.g., SEQ ID NOs:2 or 6 (TABLE 2), withor without their signal sequences and the chimeric Fc polypeptide of SEQID NO:4 (TABLE 2).

As used herein, the term “portion” when applied to a chimericpolypeptide refers to one of the components or moieties of such chimericpolypeptide (e.g., “a chimeric polypeptide comprising a FVIII portionand an Fc portion,” or “the FVIII portion of the chimeric polypeptide”).In other words, the term “portion” is used to indicate the source ofdifferent components in the chimeric polypeptide, but is not used toindicate a fragment of the FVIII protein or a fragment of an Fc region.

The chimeric polypeptide can comprise a sequence at least 90% or 95%identical to the FVIII and Fc amino acid sequence shown in TABLE 2A(i)without a signal sequence (amino acids 20 to 1684 of SEQ ID NO:2) or atleast 90% or 95% identical to the FVIII and Fc amino acid sequence shownin TABLE 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ IDNO:2), wherein the sequence has FVIII activity. The FVIII activity canbe measured by activated Partial Thromboplastin Time (aPPT) assay,chromogenic assay, or other known methods. The chimeric polypeptide cancomprise a sequence identical to the FVIII and Fc amino acid sequenceshown in TABLE 2A(i) without a signal sequence (amino acids 20 to 1684of SEQ ID NO:2) or identical to the FVIII and Fc amino acid sequenceshown in TABLE 2A(i) with a signal sequence (amino acids 1 to 1684 ofSEQ ID NO:2).

In some embodiments, the FVIII portion comprises a FVIII A3 domain. Insome embodiments, the FVIII portion comprises human FVIII. In someembodiments, the FVIII portion has a full or partial deletion of the Bdomain. In some embodiments, the FVIII portion is at least 90% or 95%identical to a FVIII amino acid sequence shown in TABLE 2 without asignal sequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20to 2351 of SEQ ID NO:6). In some embodiments, the FVIII portion isidentical to a FVIII amino acid sequence shown in TABLE 2 without asignal sequence (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 20to 2351 of SEQ ID NO:6). The FVIII portion is at least 90% or 95%identical to a FVIII amino acid sequence shown in TABLE 2 with a signalsequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351of SEQ ID NO:6). In some embodiments, the FVIII portion is identical toa FVIII amino acid sequence shown in TABLE 2 with a signal sequence(amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ IDNO:6). In some embodiments, the FVIII portion has coagulation activity.

In some embodiments, the Fc portion is identical to the Fc amino acidsequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 oramino acids 2352 to 2578 of SEQ ID NO:6). In some embodiments, thechimeric polypeptide is in the form of a hybrid comprising a secondpolypeptide in association with the chimeric polypeptide, wherein thesecond polypeptide consists essentially of or consists of the Fc portionor the FcRn binding partner.

In some embodiments, the clotting factor portion of the chimericpolypeptide comprises Factor IX. In some embodiments, the Factor IXportion of the chimeric polypeptide is at least 90%, 95%, or 100%identical to a FIX amino acid sequence shown in TABLE 2 without a signalsequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351of SEQ ID NO:6). In some embodiments, the chimeric polypeptide is amonomer dimer hybrid comprising a first chain comprising a FIX portionand the Fc portion and a second chain consisting essentially of orconsisting of a Fc portion.

In some embodiments, the chimeric polypeptide comprises a Factor VIIportion. In some embodiments, the chimeric polypeptide is a monomerdimer hybrid comprising a first chain comprising a FVII portion and theFc portion and a second chain consisting essentially of or consisting ofa Fc portion. In some embodiments, the FVII portion is inactive FVII,activated FVII, or activatable FVII.

In some embodiments, the chimeric polypeptide as disclosed hereincomprises a clotting factor other than FVIII, e.g., FVII, FVIIa, or FIX.In one example, FVII is Factor VII zymogen (inactive form of FVII),activated FVII, or activatable FVII. In another example, FIX is FIXzymogen or activated FIX. A variety of non-clotting factor portionscapable of increasing the half-life of a polypeptide are known in theart.

In some embodiments, a chimeric polypeptide comprising a FVIII portionhas an increased half-life (t1/2) over a polypeptide consisting of thesame FVIII portion without the non FVIII portion. A chimeric FVIIIpolypeptide with an increased t_(1/2) can be referred to herein as along-acting FVIII. Long-acting chimeric FVIII polypeptides include,e.g., FVIII fused to Fc (including, e.g., chimeric FVIII polypeptides inthe form of a hybrid such as a FVIIIFc monomer dimer hybrid; see Example1, FIG. 1, and Table 2A; and U.S. Pat. Nos. 7,404,956 and 7,348,004),and FVIII fused to albumin.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

“Factor VIII,” abbreviated throughout the instant application as“FVIII,” as used herein, means functional FVIII polypeptide in itsnormal role in coagulation, unless otherwise specified. Thus, the termFVIII includes variant polypeptides that are functional. FVIII proteinscan be the human, porcine, canine, and murine FVIII proteins. Asdescribed in the Background Art section, the full length polypeptide andpolynucleotide sequences are known, as are many functional fragments,mutants and modified versions. Examples of human FVIII sequences areshown as subsequences in SEQ ID NOs:2 or 6 (TABLE 2). FVIII polypeptidesinclude, e.g., full-length FVIII, full-length FVIII minus Met at theN-terminus, mature FVIII (minus the signal sequence), mature FVIII withan additional Met at the N-terminus, and/or FVIII with a full or partialdeletion of the B domain. FVIII variants include B domain deletions,whether partial or full deletions.

A great many functional FVIII variants are known, as is discussed aboveand below. In addition, hundreds of nonfunctional mutations in FVIIIhave been identified in hemophilia patients, and it has been determinedthat the effect of these mutations on FVIII function is due more towhere they lie within the 3-dimensional structure of FVIII than on thenature of the substitution (Cutler et al., Hum. Mutat. 19:274-8 (2002)),incorporated herein by reference in its entirety. In addition,comparisons between FVIII from humans and other species have identifiedconserved residues that are likely to be required for function (Cameronet al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat. No. 6,251,632),incorporated herein by reference in its entirety.

The human FVIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which isincorporated herein by reference in its entirety, and the amino acidsequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199,incorporated herein by reference in its entirety, discloses arecombinant DNA method for producing FVIII in mammalian host cells andpurification of human FVIII. Human FVIII expression in CHO (Chinesehamster ovary) cells and BHKC (baby hamster kidney cells) has beenreported. Human FVIII has been modified to delete part or all of the Bdomain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each of which isincorporated herein by reference in its entirety), and replacement ofthe human FVIII B domain with the human factor V B domain has beenperformed (U.S. Pat. No. 5,004,803, incorporated herein by reference inits entirety). The cDNA sequence encoding human FVIII and predictedamino acid sequence are shown in SEQ ID NOs:1 and 2, respectively, ofU.S. Application Publ. No. 2005/0100990, incorporated herein byreference in its entirety.

U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein by referencein its entirety, reports functional mutants of FVIII having reducedantigenicity and reduced immunoreactivity. U.S. Pat. No. 6,376,463,Lollar, J. S., incorporated herein by reference in its entirety, alsoreports mutants of FVIII having reduced immunoreactivity. U.S.Application Publ. No. 2005/0100990, Saenko et al., incorporated hereinby reference in its entirety, reports functional mutations in the A2domain of FVIII.

A number of functional FVIII molecules, including B-domain deletions,are disclosed in the following patents U.S. Pat. No. 6,316,226 and U.S.Pat. No. 6,346,513, both assigned to Baxter; U.S. Pat. No. 7,041,635assigned to In2Gen; U.S. Pat. No. 5,789,203, U.S. Pat. No. 6,060,447,U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620 assigned to Chiron;U.S. Pat. No. 5,972,885 and U.S. Pat. No. 6,048,720 assigned toBiovitrum, U.S. Pat. No. 5,543,502 and U.S. Pat. No. 5,610,278 assignedto Novo Nordisk; U.S. Pat. No. 5,171,844 assigned to Immuno Ag; U.S.Pat. No. 5,112,950 assigned to Transgene S.A.; U.S. Pat. No. 4,868,112assigned to Genetics Institute, each of which is incorporated herein byreference in its entirety.

The porcine FVIII sequence is published, (Toole, J. J., et al., Proc.Natl. Acad. Sci. USA 83:5939-5942 (1986)), incorporated herein byreference in its entirety, and the complete porcine cDNA sequenceobtained from PCR amplification of FVIII sequences from a pig spleencDNA library has been reported (Healey, J. F. et al., Blood 88:4209-4214(1996), incorporated herein by reference in its entirety). Hybridhuman/porcine FVIII having substitutions of all domains, all subunits,and specific amino acid sequences were disclosed in U.S. Pat. No.5,364,771 by Lollar and Runge, and in WO 93/20093, incorporated hereinby reference in its entirety. More recently, the nucleotide andcorresponding amino acid sequences of the A1 and A2 domains of porcineFVIII and a chimeric FVIII with porcine A1 and/or A2 domains substitutedfor the corresponding human domains were reported in WO 94/11503,incorporated herein by reference in its entirety. U.S. Pat. No.5,859,204, Lollar, J. S., also discloses the porcine cDNA and deducedamino acid sequences. U.S. Pat. No. 6,458,563, incorporated herein byreference in its entirety assigned to Emory discloses a B-domain deletedporcine FVIII.

The FVIII (or FVIII portion of a chimeric polypeptide) can be at least90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; andamino acids 20 to 2351 of SEQ ID NO:6), wherein said FVIII portion hasFVIII activity. The FVIII (or FVIII portion of a chimeric polypeptide)can be identical to a FVIII amino acid sequence shown in TABLE 2 withouta signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; and aminoacids 20 to 2351 of SEQ ID NO:6).

The FVIII (or FVIII portion of a chimeric polypeptide) can be at least90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 and aminoacids 1 to 2351 of SEQ ID NO:6), wherein said FVIII portion has FVIIIactivity. The FVIII (or FVIII portion of a chimeric polypeptide) can beidentical to a FVIII amino acid sequence shown in TABLE 2 with a signalsequence (amino acids 1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351of SEQ ID NO:6).

In some embodiments, the clotting factor is a mature form of Factor VIIzymogen, activated FVII (FVIIa), or activatable FVII, or a variantthereof. Factor VII (FVII, F7; also referred to as Factor 7, coagulationfactor VII, serum factor VII, serum prothrombin conversion accelerator,SPCA, proconvertin and eptacog alpha) is a serine protease that is partof the coagulation cascade. FVII includes a G1a domain, two EGF domains(EGF-1 and EGF-2), and a serine protease domain (or peptidase S1 domain)that is highly conserved among all members of the peptidase S1 family ofserine proteases, such as for example with chymotrypsin. FVII occurs asa single chain zymogen, an activated zymogen-like two-chain polypeptideand a fully activated two-chain form. As used herein, a “zymogen-like”protein or polypeptide refers to a protein that has been activated byproteolytic cleavage, but still exhibits properties that are associatedwith a zymogen, such as, for example, low or no activity, or aconformation that resembles the conformation of the zymogen form of theprotein. For example, when it is not bound to tissue factor, thetwo-chain activated form of FVII is a zymogen-like protein; it retains aconformation similar to the uncleaved FVII zymogen, and, thus, exhibitsvery low activity. Upon binding to tissue factor, the two-chainactivated form of FVII undergoes conformational change and acquires itsfull activity as a coagulation factor. Exemplary FVII variants includethose with increased specific activity, e.g., mutations that increasethe activity of FVII by increasing its enzymatic activity (Kcat or Km).Such variants have been described in the art and include, e.g., mutantforms of the molecule as described for example in Persson et al. 2001.PNAS 98:13583; Petrovan and Ruf 2001. J. Biol. Chem. 276:6616; Perssonet al. 2001 J. Biol. Chem. 276:29195; Soejima et al. 2001. J. Biol.Chem. 276:17229; Soejima et al. 2002. J. Biol. Chem. 247:49027. In oneembodiment, a variant form of FVII includes the mutations. Exemplarymutations include V158D-E296V-M298Q. In another embodiment, a variantform of FVII includes a replacement of amino acids 608-619(LQQSRKVGDSPN, corresponding to the 170-loop) from the FVII maturesequence with amino acids EASYPGK from the 170-loop of trypsin. Highspecific activity variants of FIX are also known in the art. Forexample, Simioni et al. (2009 N.E. Journal of Medicine 361:1671)describe an R338L mutation. Chang et al. (1988 JBC 273:12089) and Pierriet al. (2009 Human Gene Therapy 20:479) describe an R338A mutation.Other mutations are known in the art and include those described, e.g.,in Zogg and Brandstetter. 2009 Structure 17:1669; Sichler et al. 2003.J. Biol. Chem. 278:4121; and Sturzebecher et al. 1997. FEBS Lett412:295. The contents of these references are incorporated herein byreference. Full activation, which occurs upon conformational change froma zymogen-like form, occurs upon binding to is co-factor tissue factor.Also, mutations can be introduced that result in the conformation changein the absence of tissue factor. Hence, reference to FVIIa includes azymogen-like form, a fully activated two-chain form, or an activatableform. An “activatable Factor VII” is Factor VII in an inactive form(e.g., in its zymogen form) that is capable of being converted to anactive form.

In some embodiments, the clotting factor is a mature form of Factor IXor a variant thereof. Factor IX circulates as a 415 amino acid, singlechain plasma zymogen (A. Vysotchin et al., J. Biol. Chem. 268, 8436(1993)). The zymogen of FIX is activated by FXIa or by the tissuefactor/FVIIa complex. Specific cleavages between arginine-alanine145-146 and arginine-valine 180-181 result in a light chain and a heavychain linked by a single disulfide bond between cysteine 132 andcysteine 289 (S. Bajaj et al., Biochemistry 22, 4047 (1983)).

The structural organization of FIX is similar to that of the vitaminK-dependent blood clotting proteins FVII, FX and protein C (B. Furie andB. Furie, supra). The approximately 45 amino acids of the amino terminuscomprise the gamma-carboxyglutamic acid, or G1a, domain. This isfollowed by two epidermal growth factor homology domains (EGF), anactivation peptide and the catalytic “heavy chain” which is a member ofthe serine protease family (A. Vysotchin et al., J. Biol. Chem. 268,8436 (1993); S. Spitzer et al., Biochemical Journal 265, 219 (1990); H.Brandstetter et al., Proc. Natl. Acad Sci. USA 92, 9796 (1995)).

“Equivalent dose,” as used herein, means the same dose of FVIII activityas expressed in International Units, which is independent of molecularweight of the polypeptide in question. One International Unit (IU) ofFVIII activity corresponds approximately to the quantity of FVIII in onemilliliter of normal human plasma. Several assays are available formeasuring FVIII activity, including the European Pharmacopoeiachromogenic substrate assay and a one stage clotting assay.

“Fc,” as used herein, means functional neonatal Fc receptor (FcRn)binding partners, unless otherwise specified. An FcRn binding partner isany molecule that can be specifically bound by the FcRn receptor withconsequent active transport by the FcRn receptor of the FcRn bindingpartner. Thus, the term Fc includes any variants of IgG Fc that arefunctional. The region of the Fc portion of IgG that binds to the FcRnreceptor has been described based on X-ray crystallography (Burmeisteret al., Nature 372:379 (1994), incorporated herein by reference in itsentirety). The major contact area of the Fc with the FcRn is near thejunction of the CH2 and CH3 domains. Fc-FcRn contacts are all within asingle Ig heavy chain. The FcRn binding partners include, e.g., wholeIgG, the Fc fragment of IgG, and other fragments of IgG that include thecomplete binding region of FcRn. The major contact sites include aminoacid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 ofthe CH2 domain and amino acid residues 385-387, 428, and 433-436 of theCH3 domain.

References made to amino acid numbering of immunoglobulins orimmunoglobulin fragments, or regions, are all based on Kabat et al.1991, Sequences of Proteins of Immunological Interest, U. S. Departmentof Public Health, Bethesda; MD, incorporated herein by reference in itsentirety. (The FcRn receptor has been isolated from several mammalianspecies including humans. The sequences of the human FcRn, rat FcRn, andmouse FcRn are known (Story et al., J. Exp. Med. 180: 2377 (1994),incorporated herein by reference in its entirety.) An Fc can comprisethe CH2 and CH3 domains of an immunoglobulin with or without the hingeregion of the immunoglobulin. Exemplary Fc variants are provided in WO2004/101740 and WO 2006/074199, incorporated herein by reference in itsentirety.

An Fc (or Fc portion of a chimeric polypeptide) can contain one or moremutations, and combinations of mutations. E.g., an Fc (or Fc portion ofa chimeric polypeptide) can contain mutations conferring increasedhalf-life such as M252Y, S254T, T256E, and combinations thereof, asdisclosed in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which isincorporated herein by reference in its entirety; H433K, N434F, andcombinations thereof, as disclosed in Vaccaro et al., Nat. Biotechnol.23:1283 (2005), which is incorporated herein by reference in itsentirety; the mutants disclosed at pages 1-2, paragraph [0012], andExamples 9 and 10 of U.S. Application Publ. No. 2009/0264627 A1, whichis incorporated herein by reference in its entirety; and the mutantsdisclosed at page 2, paragraphs [0014] to [0021] of U.S. ApplicationPubl. No. 20090163699 A1, which is incorporated herein by reference inits entirety.

Fc (or Fc portion of a chimeric polypeptide) can also include, e.g., thefollowing mutations: The Fc region of IgG can be modified according towell recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include, e.g., modifications remotefrom the FcRn contact sites as well as modifications within the contactsites that preserve or even enhance binding to the FcRn. For example thefollowing single amino acid residues in human IgG1 Fc (Fcy1) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A,R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A,Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A,N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A,Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A,S440A, S444A, and K447A, where for example P238A represents wildtypeproline substituted by alanine at position number 238. In addition toalanine other amino acids can be substituted for the wildtype aminoacids at the positions specified above.

Mutations can be introduced singly into Fc giving rise to more than onehundred FcRn binding partners distinct from native Fc. Additionally,combinations of two, three, or more of these individual mutations can beintroduced together, giving rise to hundreds more FcRn binding partners.Certain of these mutations can confer new functionality upon the FcRnbinding partner. For example, one embodiment incorporates N297A,removing a highly conserved N-glycosylation site. The effect of thismutation is to reduce immunogenicity, thereby enhancing circulatinghalf-life of the FcRn binding partner, and to render the FcRn bindingpartner incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA,without compromising affinity for FcRn (Routledge et al. 1995,Transplantation 60:847, which is incorporated herein by reference in itsentirety; Friend et al. 1999, Transplantation 68:1632, which isincorporated herein by reference in its entirety; Shields et al. 1995,J. Biol. Chem. 276:6591, which is incorporated herein by reference inits entirety).

Additionally, at least three human Fc gamma receptors appear torecognize a binding site on IgG within the lower hinge region, generallyamino acids 234-237. Therefore, another example of new functionality andpotential decreased immunogenicity can arise from mutations of thisregion, as for example by replacing amino acids 233-236 of human IgG1“ELLG” to the corresponding sequence from IgG2 “PVA” (with one aminoacid deletion). It has been shown that FcγRI, FcγRII, and FcγRIII whichmediate various effector functions will not bind to IgG1 when suchmutations have been introduced (Ward and Ghetie, Therapeutic Immunology2:77 (1995), which is incorporated herein by reference in its entirety;and Armour et al., Eur. J. Immunol. 29:2613 (1999), which isincorporated herein by reference in its entirety). As a further exampleof new functionality arising from mutations described above affinity forFcRn can be increased beyond that of wild type in some instances. Thisincreased affinity can reflect an increased “on” rate, a decreased “off”rate or both an increased “on” rate and a decreased “off” rate.Mutations believed to impart an increased affinity for FcRn include,e.g., T256A, T307A, E380A, and N434A (Shields et al., J. Biol. Chem.276:6591 (2001), which is incorporated herein by reference in itsentirety).

The Fc (or Fc portion of a chimeric polypeptide) can be at least 90% or95% identical to the Fc amino acid sequence shown in TABLE 2 (aminoacids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ IDNO:6). The Fc (or Fc portion of a chimeric polypeptide) can be identicalto the Fc amino acid sequence shown in TABLE 2 (amino acids 1458 to 1684of SEQ ID NO:2 and amino acids 2352 to 2578 of SEQ ID NO:6).

“Hybrid” polypeptides and proteins, as used herein, means a combinationof a chimeric polypeptide with a second polypeptide. The chimericpolypeptide and the second polypeptide in a hybrid can be associatedwith each other via protein-protein interactions, such as charge-chargeor hydrophobic interactions. The chimeric polypeptide and the secondpolypeptide in a hybrid can be associated with each other via disulfideor other covalent bond(s). Hybrids are described in WO 2004/101740 andWO 2006/074199, each of which is incorporated herein by reference in itsentirety. See also U.S. Pat. Nos. 7,404,956 and 7,348,004, each of whichis incorporated herein by reference in its entirety. The secondpolypeptide can be a second copy of the same chimeric polypeptide or itcan be a non-identical chimeric polypeptide. See, e.g., FIG. 1, Example1, and TABLE 2.

In one embodiment, the second polypeptide is a polypeptide comprising anFc. In another embodiment, the chimeric polypeptide is a chimericFVIII-Fc polypeptide and the second polypeptide consists essentially ofFc, e.g., the hybrid polypeptide of Example 1, which is a rFVIIIFcrecombinant fusion protein consisting of a single molecule ofrecombinant B-domain deleted human FVIII (BDD-rFVIII) fused to thedimeric Fc domain of the human IgG1, with no intervening linkersequence. This hybrid polypeptide is referred to herein as FVIIIFcmonomeric Fc fusion protein, FVIIIFc monomer hybrid, monomeric FVIIIIFchybrid, and FVIIIFc monomer-dimer. See Example 1, FIG. 1, and TABLE 2A.The Examples provide preclinical and clinical data for this hybridpolypeptide.

The second polypeptide in a hybrid can comprise or consist essentiallyof a sequence at least 90% or 95% identical to the amino acid sequenceshown in TABLE 2A(ii) without a signal sequence (amino acids 21 to 247of SEQ ID NO:4) or at least 90% or 95% identical to the amino acidsequence shown in TABLE 2A(ii) with a signal sequence (amino acids 1 to247 of SEQ ID NO-:4). The second polypeptide can comprise or consistessentially of a sequence identical to the amino acid sequence shown inTABLE 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ IDNO:4) or identical to the amino acid sequence shown in TABLE 2A(ii) witha signal sequence (amino acids 1 to 247 of SEQ ID NO:4).

FIG. 1 is a schematic showing the structure of a B domain deletedFVIII-Fc chimeric polypeptide, and its association with a secondpolypeptide that is an Fc polypeptide. To obtain this hybrid, the codingsequence of human recombinant B-domain deleted FVIII was obtained byreverse transcription-polymerase chain reaction (RT-PCR) from humanliver poly A RNA (Clontech) using FVIII-specific primers. The FVIIIsequence includes the native signal sequence for FVIII. The B-domaindeletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638;4969 bp) for a total deletion of 2682 bp. Then, the coding sequence forhuman recombinant Fc was obtained by RT-PCR from a human leukocyte cDNAlibrary (Clontech) using Fc specific primers. Primers were designed suchthat the B-domain deleted FVIII sequence was fused directly to theN-terminus of the Fc sequence with no intervening linker. The FVIIIFcDNA sequence was cloned into the mammalian dual expression vectorpBUDCE4.1 (Invitrogen) under control of the CMV promoter. A secondidentical Fc sequence including the mouse Igk signal sequence wasobtained by RT-PCR and cloned downstream of the second promoter, EF1α,in the expression vector pBUDCE4.1.

The rFVIIIFc expression vector was transfected into human embryonickidney 293 cells (HEK293H; Invitrogen) using Lipofectamine 2000transfection reagent (Invitrogen). Stable clonal cell lines weregenerated by selection with Zeocin (Invitrogen). One clonal cell line,3C4-22 was used to generate FVIIIFc for characterization in vivo.Recombinant FVIIIFc was produced and purified (McCue et al. 2009) atBiogen Idec (Cambridge, Mass.). The transfection strategy describedabove was expected to yield three products, i.e., monomeric rFVIIIFchybrids, dimeric rFVIIIFc hybrids and dimeric Fc. However, there wasessentially no dimeric rFVIIIFc detected in the conditioned medium fromthese cells. Rather, the conditioned medium contained Fc and monomericrFVIIIFc. It is possible that the size of dimeric rFVIIIFc was too greatand prevented efficient secretion from the cell. This result wasbeneficial since it rendered the purification of the monomer lesscomplicated than if all three proteins had been present. The materialused in these studies had a specific activity of approximately 9000IU/mg.

“Dosing interval,” as used herein, means the dose of time that elapsesbetween multiple doses being administered to a subject. The comparisonof dosing interval can be carried out in a single subject or in apopulation of subjects and then the average obtained in the populationcan be calculated.

The dosing interval when administering a chimeric FVIII polypeptide,e.g., a chimeric FVIII-Fc polypeptide (a polypeptide comprising a FVIIIor a hybrid) of the present disclosure can be at least about one andone-half times longer than the dosing interval required for anequivalent dose of said FVIII without the non-FVIII portion, e.g.,without the Fc portion (a polypeptide consisting of said FVIII). Thedosing interval can be at least about one and one-half to six timeslonger, one and one-half to five times longer, one and one-half to fourtimes longer, one and one-half to three times longer, or one andone-half to two times longer, than the dosing interval required for anequivalent dose of said FVIII without the non-FVIII portion, e.g.,without the Fc portion (a polypeptide consisting of said FVIII).

The dosing interval can be at least about one and one-half, two, two andone-half, three, three and one-half, four, four and one-half, five, fiveand one-half or six times longer than the dosing interval required foran equivalent dose of said FVIII without the non-FVIII portion, e.g.,without the Fc portion (a polypeptide consisting of said FVIII). Thedosing interval can be about every three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, or fourteen days or longer. Thedosing interval can be at least about one and one-half to 5, one andone-half, 2, 3, 4, or 5 days or longer. For on-demand treatment, thedosing interval of said chimeric polypeptide or hybrid is about onceevery 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or longer.

In one embodiment, the effective dose is 25-65 IU/kg (25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 62, 64,or 65 IU/kg) and the dosing interval is once every 3-5, 3-6, 3-7, 3, 4,5, 6, 7, or 8 or more days, or three times per week, or no more thanthree times per week. In another embodiment, the effective dose is 65IU/kg and the dosing interval is once weekly, or once every 6-7 days.The doses can be administered repeatedly as long as they are necessary(e.g., at least 10, 20, 28, 30, 40, 50, 52, or 57 weeks, at least 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 years).

In certain embodiments, the effective dose for on-demand treatment is20-50 IU/Kg (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50IU/kg). The on-demand treatment can be one time dosing or repeateddosing. For repeated dosing, the dosing interval can be every 12-24hours, every 24-36 hours, every 24-48 hours, every 36-48 hours, or every48-72 hours. Accordingly, the term “repeated dosing” refers to theadministration of more than one dose over a period of time. Dosesadministered under a “repeated dosing” regimen are referred to as“repeated doses.” In some embodiments, each of the repeated doses isseparated from another by at least about 12 hours, at least about 24hours, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about seven days, at least about eight days, at least about ninedays, at least about ten days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, or at least about15 days. In some embodiments, the repeated doses comprise at least abouttwo doses, at least about five doses, at least about 10 doses, at leastabout 20 doses, at least about 25 doses, at least about 30 doses, atleast about 35 doses, at least about 40 doses, at least about 45 doses,at least about 50 doses, at least about 55 doses, at least about 60doses, at least about 65 doses, or at least about 70 doses. the repeateddoses comprise from about two doses to about 100 doses, from about fivedoses to about 80 doses, from about 10 doses to about 70 doses, fromabout 10 doses to about 60 doses, from about 10 doses to about 50 doses,from about 15 doses to about 40 doses, from about 15 doses to about 30doses, from about 20 doses to about 30 doses, or from about 20 doses toabout 40 doses. the repeated doses comprise about two doses, about fivedoses, about 10 doses, about 15 doses, about 20 doses, about 25 doses,about 30 doses, about 35 doses, about 40 doses, about 45 doses, about 50doses, about 55 doses, about 60 doses, about 65 does, about 70 doses,about 75 doses, about 80 doses, about 90 doses, or about 100 doses.

In some embodiments, the subject is further administered, after therepeated doses, a pharmaceutical composition comprising a clottingfactor protein which comprises the clotting factor, but does notcomprise an Fc portion. This clotting factor can be a full length ormature clotting factor. In some embodiments, such clotting factor can beADVATE®, RECOMBINATE®, KOGENATE FS®, HELIXATE FS®, XYNTHA®/REFACTO ABC),HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®, ALPHANATE®, KOATE-DVI®,AND HYATE:C®

The terms “long-acting” and “long-lasting” are used interchangeablyherein. “Long-lasting clotting factors” or “long-acting clottingfactors” (e.g., long-acting FVII, long-acting FVIII, and long-actingFIX) are clotting factors, e.g., FVII, FVIII, or FIX, having anincreased half-life (also referred to herein as t_(1/2), t_(1/2) beta,elimination half-life and HL) over a reference clotting factor, e.g.,FVII, FVIII or FIX. The “longer” FVIII activity can be measured by anyknown methods in the art, e.g., aPTT assay, chromogenic assay, ROTEM,TGA, and etc. In one embodiment, the “longer” FVIII activity can beshown by the T_(1/2)beta (activity). In another embodiment, the “longer”FVIII activity can be shown by the level of FVIII antigen present inplasma, e.g., by the T_(1/2)beta (antigen). In other embodiments, thelong-acting or long-lasting FVIII polypeptide works longer in acoagulation cascade, e.g., is active for a longer period, compared to awild-type FVIII polypeptide, REFACTO® or ADVATE®.

The increased half-life of a long-acting clotting factor, e.g., along-acting FVIII, can be due to fusion to one or more non-clottingfactor polypeptides such as, e.g., Fc. The increased half-life can bedue to one or more modification, such as, e.g., pegylation. Exemplarylong-acting clotting factor (e.g., long-acting FVIII polypeptides)include, e.g., chimeric clotting factors (e.g., chimeric FVIIIpolypeptides comprising Fc), and chimeric clotting factors comprisingalbumin (e.g., chimeric FVIII polypeptides comprising albumin).Additional exemplary long-acting clotting factors (e.g., long-actingFVIII polypeptides) include, e.g., pegylated clotting factors (e.g.,pegylated FVIII).

The “reference” polypeptide, e.g., in the case of a long-acting chimericclotting factor (e.g., a long-acting chimeric FVIII polypeptide), is apolypeptide consisting essentially of the clotting factor portion of thechimeric polypeptide. For example, in the case of a long-acting FVIII,the reference polypeptide is the FVIII portion of the chimericpolypeptide, e.g., the same FVIII portion without the Fc portion, orwithout the albumin portion. Likewise, the reference polypeptide in thecase of a modified FVIII is the same FVIII without the modification,e.g., a FVIII without the pegylation.

In some embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a subject:

-   -   a mean residence time (MRT) (activity) in said subject of about        14-41.3 hours;    -   a clearance (CL) (activity) in said subject of about 1.22-5.19        mL/hour/kg or less;    -   a t_(1/2)beta (activity) in said subject of about 11-26.4 hours;    -   an incremental recovery (K value) (activity; observed) in said        subject of about 1.38-2.88 IU/dL per IU/kg;    -   a V_(ss) (activity) in said subject of about 37.7-79.4 mL/kg;        and    -   an AUC/dose in said subject of about 19.2-81.7 IU*h/dL per        IU/kg.

In some embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

-   -   a mean incremental recovery (K-Value) (activity; observed)        greater that 1.38 IU/dL per IU/kg;    -   a mean incremental recovery (K-Value) (activity; observed) of at        least about 1.5, at least about 1.85, or at least about 2.46        IU/dL per IU/kg.    -   a mean clearance (CL) (activity) in said patient population of        about 2.33±1.08 mL/hour/kg or less;    -   a mean clearance (CL) (activity) in said patient population of        about 1.8-2.69 mL/hour/kg;    -   a mean clearance (CL) (activity) in said patient population that        is about 65% of the clearance of a polypeptide comprising said        FVIII without modification;    -   a mean residence time (MRT) (activity) in said patient        population of at least about 26.3±8.33 hours;    -   a mean MRT (activity) in said patient population of about        25.9-26.5 hours;    -   a mean MRT (activity) in said patent population that is about        1.5 fold longer than the mean MRT of a polypeptide comprising        said FVIII without modification;    -   a mean t_(1/2)beta (activity) in said patient population of        about 18.3±5.79 hours;    -   a mean t_(1/2)beta (activity) in said patient population that is        about 18-18.4 hours;    -   a mean t_(1/2)beta (activity) in said patient population that is        about 1.5 fold longer than the mean t_(1/2)beta of a polypeptide        comprising said FVIII without modification;    -   a mean incremental recovery (K value) (activity; observed) in        said patient population of about 2.01±0.44 IU/dL per IU/kg;    -   a mean incremental recovery (K value) (activity; observed) in        said patient population of about 1.85-2.46 IU/dL per IU/kg;    -   a mean incremental recovery (K value) (activity; observed) in        said patient population that is about 90% of the mean        incremental recovery of a polypeptide comprising said FVIII        without modification;    -   a mean V_(ss) (activity) in said patient population of about        55.1±12.3 mL/kg;    -   a mean V_(ss) (activity) in said patient population of about        45.3-56.1 mL/kg;    -   a mean AUC/dose (activity) in said patient population of about        49.9±18.2 IU*h/dL per IU/kg;    -   a mean AUC/dose (activity) in said patient population of about        44.8-57.6 IU*h/dL per IU/kg.

In other embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

a C_(max) _(_)OBS in said subject administered with the chimericpolypeptide is comparable to the C_(max) _(_)OBS in a subjectadministered with the same amount of a polypeptide consisting of thefull-length, mature FVIII when measured by a one stage (aPTT) assay or atwo stage (chromogenic) assay;

a C_(max) _(_)OBS in said subject of about 60.5 IU/dL, about 60.5±1IU/dL, about 60.5±2 IU/dL, about 60.5±3 IU/dL, about 60.5±4 IU/dL, about60.5±5 IU/dL, about 60.5±6 IU/dL, about 60.5±7 IU/dL, about 60.5±8IU/dL, about 60.5±9 IU/dL, or about 60.5±10 IU/dL as measured by a onestage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide isadministered;

a C_(max) _(_)OBS in said subject of about 53.1-69 IU/dL as measured bya one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptideis administered;

a C_(max) _(_)OBS in said subject of about 119 IU/dL, about 119±1 IU/dL,about 119±2 IU/dL, about 119±3 IU/dL, about 119±4 IU/dL, about 119±5IU/dL, about 119±6 IU/dL, about 119±7 IU/dL, about 119±8 IU/dL, about119±9 IU/dL, about 119±10 IU/dL, about 119±11 IU/dL, about 119±12 IU/dL,about 119±13 IU/dL, about 119±14 IU/dL, about 119±15 IU/dL, about 119±16IU/dL, about 119±17 IU/dL, or about 119±18 IU/dL, as measured by a onestage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide isadministered;

a C_(max) _(_)OBS in said subject of about 103-136 IU/dL as measured bya one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptideis administered;

a C_(max) _(_)OBS in said subject of about 76.5 IU/dL, about 76.5±1IU/dL, about 76.5±2 IU/dL, about 76.5±3 IU/dL, about 76.5±4 IU/dL, about76.5±5 IU/dL, about 76.5±6 IU/dL, about 76.5±7 IU/dL, about 76.5±8IU/dL, about 76.5±9 IU/dL, about 76.5±10 IU/dL, about 76.5±11 IU/dL,about 76.5±12 IU/dL, about 76.5±13 IU/dL, about 76.5±14 IU/dL, or about76.5±15 IU/dL, as measured by a two stage (chromogenic) assay when about25 IU/kg of the chimeric polypeptide is administered;

a C_(max) _(_)OBS in said subject of about 64.9-90.1 IU/dL as measuredby a two stage (chromogenic) assay when about 25 IU/kg of the chimericpolypeptide is administered;

a C_(max) _(_)OBS in said subject of about 182 IU/dL, about 182±2 IU/dL,about 182±4 IU/dL, about 182±6 IU/dL, about 182±8 IU/dL, about 182±10IU/dL, about 182±12 IU/dL, about 182±14 IU/dL, about 182±16 IU/dL, about182±18 IU/dL, or about 182±20 IU/dL as measured by a two stage(chromogenic) assay when about 65 IU/kg of the chimeric polypeptide isadministered; or

a C_(max) _(_)OBS in said subject of about 146-227 IU/dL, about 146±5IU/dL, about 146±10 IU/dL, about 227±5 IU/dL, or about 146±10 IU/dL asmeasured by a two stage (chromogenic) assay when about 65 IU/kg of thechimeric polypeptide is administered.

In certain embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

a t_(1/2)beta (activity) in said subject that is at least 1.48, 1.49,1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61,1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73,1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85,1.86, 1.87, 1.88, 1.89, or 1.90 times higher than the t_(1/2)beta(activity) in a subject administered with the same amount of apolypeptide consisting of the full-length, mature FVIII when measured bya one stage (aPTT) assay or a two stage (chromogenic) assay;

a t_(1/2)beta (activity) in said subject of about 18.8 hours, 18.8±1hours, 18.8±1 hours, 18.8±2 hours, 18.8±3 hours, 18.8±4 hours, 18.8±5hours, 18.8±6 hours, 18.8±7 hours, 18.8±8 hours, 18.8±9 hours, 18.8±10hours, or 18.8±11 hours as measured by a one stage (aPTT) assay;

a t_(1/2)beta (activity) in said subject of about 14.3-24.5 hours asmeasured by a one stage (aPTT) assay;

a t_(1/2)beta (activity) in said subject of about 16.7 hours, 16.7±1hours, 16.7±2 hours, 16.7±3 hours, 16.7±4 hours, 16.7±5 hours, 16.7±6hours, 16.7±7 hours, 16.7±8 hours, 16.7±9 hours, 16.7±10 hours, or16.7±11 hours as measured by a two stage (chromogenic) assay;

a t_(1/2)beta (activity) in said subject of about 13.8-20.1 hours asmeasured by a two stage (chromogenic) assay;

a t_(1/2)beta (activity) in said subject of about 19.8 hours, 19.8±1hours, 19.8±2 hours, 19.8±3 hours, 19.8±4 hours, 19.8±5 hours, 19.8±6hours, 19.8±7 hours, 19.8±8 hours, 19.8±9 hours, 19.8±10 hours, or19.8±11 hours as measured by a two stage (chromogenic) assay; or

a t_(1/2)beta (activity) in said subject of about 14.3-27.5 hours asmeasured by a two stage (chromogenic) assay.

In certain embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

a clearance (CL) (activity) in said subject is 0.51, 0.52, 0.53, 0.54,0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66,0.67, 0.68, 0.69, or 0.70 times lower than the clearance in a subjectadministered with the same amount of a polypeptide consisting of thefull-length, mature FVIII when measured by a one stage (aPTT) assay or atwo stage (chromogenic) assay;

a clearance (CL) (activity) in said subject of about 1.68 mL/hour/kg,1.68±0.1 mL/hour/kg, 1.68±0.2 mL/hour/kg, 1.68±0.3 mL/hour/kg, 1.68±0.4mL/hour/kg, 1.68±0.5 mL/hour/kg, 1.68±0.6 mL/hour/kg, or 1.68±0.7mL/hour/kg, as measured by a one stage (aPTT) assay when about 25 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.31-2.15mL/hour/kg as measured by a one stage (aPTT) assay when about 25 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 2.32 mL/hour/kg,2.32±0.1 mL/hour/kg, 2.32±0.2 mL/hour/kg, 2.32±0.3 mL/hour/kg, 2.32±0.4mL/hour/kg, 2.32±0.5 mL/hour/kg, 2.32±0.6 mL/hour/kg, or 2.32±0.7mL/hour/kg as measured by a one stage (aPTT) assay when about 65 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.64-3.29mL/hour/kg as measured by a one stage (aPTT) assay when about 65 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.49 mL/hour/kg,1.49±0.1 mL/hour/kg, 1.49±0.2 mL/hour/kg, 1.49±0.3 mL/hour/kg, 1.49±0.4mL/hour/kg, 1.49±0.5 mL/hour/kg, 1.49±0.6 mL/hour/kg, or 1.49±0.7mL/hour/kg as measured by a two stage (chromogenic) assay when about 25IU/kg of the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.16-1.92mL/hour/kg as measured by a two stage (chromogenic) assay when about 25IU/kg of the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.52 mL/hour/kg,1.52±0.1 mL/hour/kg, 1.52±0.2 mL/hour/kg, 1.52±0.3 mL/hour/kg, 1.52±0.4mL/hour/kg, 1.52±0.5 mL/hour/kg, 1.52±0.6 mL/hour/kg, or 1.52±0.7mL/hour/kg as measured by a two stage (chromogenic) assay when about 65IU/kg of the chimeric polypeptide is administered; or

a clearance (CL) (activity) in said subject of about 1.05-2.20mL/hour/kg as measured by a two stage (chromogenic) assay when about 65IU/kg of the chimeric polypeptide is administered.

In some embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

a MRT in said subject is at least 1.46, 1.47, 1.48, 1.49, 1.50, 1.51,1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63,1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75,1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87,1.88, 1.89, 1.90, 1.91, 1.92, or 1.93 times higher than the MRT in asubject administered with the same amount of a polypeptide consisting ofthe full-length, mature FVIII when measured by a one stage (aPTT) assayor a two stage (chromogenic) assay;

a MRT (activity) in said subject of about 27 hours, 27±1 hours, 27±2hours, 27±3 hours, 27±4 hours, 27±5 hours, 27±6 hours, 27±7 hours, 27±8hours, 27±9 hours, or 27±10 hours as measured by a one stage (aPTT)assay;

a MRT (activity) in said subject of about 20.6-35.3 hours as measured bya one stage (aPTT) assay;

a MRT (activity) in said subject of about 23.9-28.5 hours as measured bya two stage (chromogenic) assay;

a MRT (activity) in said subject of about 19.8-28.9 hours as measured bya two stage (chromogenic) assay; or

a MRT (activity) in said subject of about 20.5-39.6 hours as measured bya two stage (chromogenic) assay.

In other embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

an incremental recovery in said subject that is comparable to theIncremental Recovery in a subject administered with the same amount of apolypeptide consisting of the full-length, mature FVIII when measured bya one stage (aPTT) assay or a two stage (chromogenic) assay;

an incremental recovery in said subject of about 2.44 IU/dL per IU/kg,2.44±0.1 IU/dL per IU/kg, 2.44±0.2 IU/dL per IU/kg, 2.44±0.3 IU/dL perIU/kg, 2.44±0.4 IU/dL per IU/kg, 2.44±0.5 IU/dL per IU/kg, 2.44±0.6IU/dL per IU/kg, 2.44±0.7 IU/dL per IU/kg, 2.44±0.8 IU/dL per IU/kg,2.44±0.9 IU/dL per IU/kg, 2.44±1.0 IU/dL per IU/kg, 2.44±1.1 IU/dL perIU/kg, or 2.44±1.2 IU/dL per IU/kg as measured by a one stage (aPTT)assay when about 25 IU/kg of the chimeric polypeptide is administered;

an incremental recovery in said subject of about 2.12-2.81 IU/dL perIU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of thechimeric polypeptide is administered;

an incremental recovery in said subject of about 1.83 IU/dL per IU/kg,1.83±0.1 IU/dL per IU/kg, 1.83±0.2 IU/dL per IU/kg, 1.83±0.3 IU/dL perIU/kg, 1.83±0.4 IU/dL per IU/kg, 1.83±0.5 IU/dL per IU/kg, 1.83±0.6IU/dL per IU/kg, 1.83±0.7 IU/dL per IU/kg, 1.83±0.8 IU/dL per IU/kg,1.83±0.9 IU/dL per IU/kg, 1.83±1.0 IU/dL per IU/kg, or 1.83±1.1 IU/dLper IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg ofthe chimeric polypeptide is administered;

an incremental recovery in said subject of about 1.59-2.10 IU/dL perIU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of thechimeric polypeptide is administered;

an incremental recovery in said subject of about 3.09 IU/dL per IU/kg,3.09±0.1 IU/dL per IU/kg, 3.09±0.2 IU/dL per IU/kg, 3.09±0.3 IU/dL perIU/kg, 3.09±0.4 IU/dL per IU/kg, 3.09±0.5 IU/dL per IU/kg, 3.09±0.6IU/dL per IU/kg, 3.09±0.7 IU/dL per IU/kg, 3.09±0.8 IU/dL per IU/kg,3.09±0.9 IU/dL per IU/kg, 3.09±1.0 IU/dL per IU/kg, 3.09±1.1 IU/dL perIU/kg, 3.09±1.2 IU/dL per IU/kg, or 3.09±1.3 IU/dL per IU/kg, asmeasured by a two stage (chromogenic) assay when about 25 IU/kg of thechimeric polypeptide is administered;

an incremental recovery in said subject of about 2.80 IU/dL per IU/kg,2.80±0.1 IU/dL per IU/kg, 2.80±0.2 IU/dL per IU/kg, 2.80±0.3 IU/dL perIU/kg, 2.80±0.4 IU/dL per IU/kg, 2.80±0.5 IU/dL per IU/kg, 2.80±0.6IU/dL per IU/kg, 2.80±0.7 IU/dL per IU/kg, 2.80±0.8 IU/dL per IU/kg,2.80±0.9 IU/dL per IU/kg, 2.80±1.0 IU/dL per IU/kg, 2.80±1.1 IU/dL perIU/kg, or 2.80±1.2 IU/dL per IU/kg, as measured by a two stage(chromogenic) assay when about 65 IU/kg of the chimeric polypeptide isadministered;

an incremental recovery in said subject of about 2.61-3.66 IU/dL perIU/kg as measured by a two stage (chromogenic) assay when about 25 IU/kgof the chimeric polypeptide is administered; or

an incremental recovery in said subject of about 2.24-3.50 IU/dL perIU/kg as measured by a two stage (chromogenic) assay when about 65 IU/kgof the chimeric polypeptide is administered.

In still other embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

a V_(ss) (activity) in said subject that is comparable to the Vss(activity) in a subject administered with the same amount of apolypeptide consisting of the full-length, mature FVIII when measured bya one stage (aPTT) assay or a two stage (chromogenic) assay;

a V_(ss) (activity) in said subject of about 45.5 mL/kg, 45.5±1 mL/kg,45.5±2 mL/kg, 45.5±3 mL/kg, 45.5±4 mL/kg, 45.5±5 mL/kg, 45.5±6 mL/kg,45.5±7 mL/kg, 45.5±8 mL/kg, 45.5±9 mL/kg, 45.5±10 mL/kg, or 45.5±11mL/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg ofthe chimeric polypeptide is administered;

a V_(ss) (activity) in said subject of about 39.3-52.5 mL/kg as measuredby a one stage (aPTT) assay when about 25 IU/kg of the chimericpolypeptide is administered;

a V_(ss) (activity) in said subject of about 62.8 mL/kg, 62.8±1 mL/kg,62.8±2 mL/kg, 62.8±3 mL/kg, 62.8±4 mL/kg, 62.8±5 mL/kg, 62.8±6 mL/kg,62.8±7 mL/kg, 62.8±8 mL/kg, 62.8±9 mL/kg, 62.8±10 mL/kg, 62.8±11 mL/kg,62.8±12 mL/kg, 62.8±13 mL/kg, 62.8±14 mL/kg, 62.8±15 mL/kg, or 62.8±16mL/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of thechimeric polypeptide is administered;

a V_(ss) (activity) in said subject of about 55.2-71.5 mL/kg as measuredby a one stage (aPTT) assay when about 65 IU/kg of the chimericpolypeptide is administered;

a V_(ss) (activity) in said subject of about 35.9 mL/kg, 35.9±1 mL/kg,35.9±2 mL/kg, 35.9±3 mL/kg, 35.9±4 mL/kg, 35.9±5 mL/kg, 35.9±6 mL/kg,35.9±7 mL/kg, 35.9±8 mL/kg, 35.9±9 mL/kg, 35.9±10 mL/kg, 35.9±11 mL/kg,35.9±12 mL/kg, or 35.9±13 mL/kg, as measured by a two stage(chromogenic) assay when about 25 IU/kg of the chimeric polypeptide isadministered;

a V_(ss) (activity) in said subject of about 30.4-42.3 mL/kg as measuredby a two stage (chromogenic) assay when about 25 IU/kg of the chimericpolypeptide is administered;

a V_(ss) (activity) in said subject of about 43.4 mL/kg, 43.4±1 mL/kg,43.4±2 mL/kg, 43.4±3 mL/kg, 43.4±4 mL/kg, 43.4±5 mL/kg, 43.4±6 mL/kg,43.4±7 mL/kg, 43.4±8 mL/kg, 43.4±9 mL/kg, 43.4±10 mL/kg, 43.4±11 mL/kg,43.4±12 mL/kg, 43.4±13 mL/kg, 43.4±14 mL/kg, 43.4±15 mL/kg, or 43.4±16mL/kg, as measured by a two stage (chromogenic) assay when about 65IU/kg of the chimeric polypeptide is administered; or

a V_(ss) (activity) in said subject of about 38.2-49.2 mL/kg as measuredby a two stage (chromogenic) assay when about 65 IU/kg of the chimericpolypeptide is administered.

In yet other embodiments, the long-acting FVIII has one or more of thefollowing properties when administered to a patient population:

an AUC_(INF) in said subject that is at least 1.45, 1.46, 1.47, 1.48,1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60,1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72,1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84,1.85, 1.86, 1.87, 1.88, 1.89, 1.90 times higher than the AUC_(INF) in asubject administered with the same amount of a polypeptide consisting ofthe full-length, mature FVIII when measured by a one stage (aPTT) assayor a two stage (chromogenic) assay;

an AUC_(INF) in said subject of about 1440±316 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 25 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 1160-1880 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 25 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 1480 hr*IU/dL per IU/kg, 1480±100hr*IU/dL per IU/kg, 1480±200 hr*IU/dL per IU/kg, 1480±300 hr*IU/dL perIU/kg, 1480±400 hr*IU/dL per IU/kg, 1480±500 hr*IU/dL per IU/kg,1480±600 hr*IU/dL per IU/kg, 1480±700 hr*IU/dL per IU/kg, 1480±800hr*IU/dL per IU/kg, 1480±900 hr*IU/dL per IU/kg, or 1480±1000 hr*IU/dLper IU/kg, as measured by a one stage (aPTT) assay when about 25 IU/kgof the chimeric polypeptide is administered;

an AUC_(INF) in said subject of about 2910±1320 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 65 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 1980-3970 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 65 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 2800 hr*IU/dL per IU/kg, 2800±100hr*IU/dL per IU/kg, 2800±200 hr*IU/dL per IU/kg, 2800±300 hr*IU/dL perIU/kg, 2800±400 hr*IU/dL per IU/kg, 2800±500 hr*IU/dL per IU/kg,2800±600 hr*IU/dL per IU/kg, 2800±700 hr*IU/dL per IU/kg, 2800±800hr*IU/dL per IU/kg, 2800±900 hr*IU/dL per IU/kg, or 2800±1000 hr*IU/dLper IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg ofthe chimeric polypeptide is administered;

an AUC_(INF) in said subject of about 1660 hr*IU/dL per IU/kg, 1660±100hr*IU/dL per IU/kg, 1660±200 hr*IU/dL per IU/kg, 1660±300 hr*IU/dL perIU/kg, 1660±400 hr*IU/dL per IU/kg, 1660±500 hr*IU/dL per IU/kg,1660±600 hr*IU/dL per IU/kg, 1660±700 hr*IU/dL per IU/kg, 1660±800hr*IU/dL per IU/kg, 1660±900 hr*IU/dL per IU/kg, or 1660±1000 hr*IU/dLper IU/kg as measured by a two stage (chromogenic) assay when about 25IU/kg of the chimeric polypeptide is administered;

an AUC_(INF) in said subject of about 1300-2120 hr*IU/dL per IU/kg asmeasured by a two stage (chromogenic) assay when about 25 IU/kg of thechimeric polypeptide is administered;

an AUC_(INF) in said subject of about 4280 hr*IU/dL per IU/kg, 4280±100hr*IU/dL per IU/kg, 4280±200 hr*IU/dL per IU/kg, 4280±300 hr*IU/dL perIU/kg, 4280±400 hr*IU/dL per IU/kg, 4280±500 hr*IU/dL per IU/kg,4280±600 hr*IU/dL per IU/kg, 4280±700 hr*IU/dL per IU/kg, 4280±800hr*IU/dL per IU/kg, 4280±900 hr*IU/dL per IU/kg, 4280±1000 hr*IU/dL perIU/kg, 4280±1100 hr*IU/dL per IU/kg, 4280±1200 hr*IU/dL per IU/kg,4280±1300 hr*IU/dL per IU/kg, 4280±1400 hr*IU/dL per IU/kg, 4280±1500hr*IU/dL per IU/kg, or 4280±1600 hr*IU/dL per IU/kg as measured by a twostage (chromogenic) assay when about 65 IU/kg of the chimericpolypeptide is administered; or

an AUC_(INF) in said subject of about 2960-6190 hr*IU/dL per IU/kg asmeasured by a two stage (chromogenic) assay when about 65 IU/kg of thechimeric polypeptide is administered.

“On-demand treatment,” as used herein, means treatment that is intendedto take place over a short course of time and is in response to anexisting condition, such as a bleeding episode, or a perceived need suchas planned surgery. The terms “on-demand treatment” and “episodictreatment” are used interchangeably. Conditions that can requireon-demand (episodic) treatment include, e.g., a bleeding episode,hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage intomuscles, oral hemorrhage, trauma, trauma capitis, gastrointestinalbleeding, intracranial hemorrhage, intra-abdominal hemorrhage,intrathoracic hemorrhage, bone fracture, central nervous systembleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, or bleeding in the illiopsoas sheath. The subjectcan be in need of surgical prophylaxis, peri-operative management, ortreatment for surgery. Such surgeries include, e.g., minor surgery,major surgery, tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, total knee replacement, craniotomy, osteosynthesis, traumasurgery, intracranial surgery, intra-abdominal surgery, intrathoracicsurgery, or joint replacement surgery.

In one embodiment, on-demand (episodic) treatment resolves greater than80% (greater than 80%, greater than 81%, greater than 82%, greater than83%, greater than 84%, greater than 85%, greater than 86%, greater than87%, greater than 88%, greater than 89%, greater than 90%, greater than91%, greater than 92%, greater than 93%, greater than 94%, greater than95%, greater than 96%, greater than 97%, greater than 98%, greater than99%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% ofbleeds (e.g., spontaneous bleeds) in a single dose. In anotherembodiment, greater than 80% (greater than 81%, greater than 82%,greater than 83%, greater than 84%, greater than 85%, greater than 86%,greater than 87%, greater than 88%, greater than 89%, greater than 90%,greater than 91%, greater than 92%, greater than 93%, greater than 94%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% ofbleeding episodes are rated excellent or good by physicians afteron-demand (episodic) treatment. In other embodiments, greater than 5%,(greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, greater than 12%, greater than 13%,greater than 14%, greater than 15%, greater than 16%, greater than 17%,greater than 18%, greater than 19%, greater than 20%), or 5-20%, 5-15%,5-10%, 10-20%, or 10-15% of bleeding episodes are rated as fair byphysicians after on-demand (episodic) treatment.

“Polypeptide,” “peptide” and “protein” are used interchangeably andrefer to a polymeric compound comprised of covalently linked amino acidresidues.

“Polynucleotide” and “nucleic acid” are used interchangeably and referto a polymeric compound comprised of covalently linked nucleotideresidues. Polynucleotides can be DNA, cDNA, RNA, single stranded, ordouble stranded, vectors, plasmids, phage, or viruses. Polynucleotidesinclude, e.g., those in TABLE 1, which encode the polypeptides of TABLE2 (see TABLE 1). Polynucleotides also include, e.g., fragments of thepolynucleotides of TABLE 1, e.g., those that encode fragments of thepolypeptides of TABLE 2, such as the FVIII, Fc, signal sequence, 6Hisand other fragments of the polypeptides of TABLE 2.

“Prophylactic treatment,” as used herein, means administering a FVIIIpolypeptide in multiple doses to a subject over a course of time toincrease the level of FVIII activity in a subject's plasma. Theincreased level can be sufficient to decrease the incidence ofspontaneous bleeding or to prevent bleeding, e.g., in the event of anunforeseen injury. During prophylactic treatment, the plasma proteinlevel in the subject can not fall below the baseline level for thatsubject, or below the level of FVIII that characterizes severehemophilia (<1 IU/dl [1%]).

In one embodiment, the prophylaxis regimen is “tailored” to theindividual patient, for example, by determining PK data for each patientand administering FVIII of the present disclosure at a dosing intervalthat maintains a trough level of 1-3% FVIII activity. Adjustments can bemade when a subject experiences unacceptable bleeding episodes definedas ≥2 spontaneous bleeding episodes over a rolling two-month period. Inthis case, adjustment will target trough levels of 3-5%. In anotherembodiment, prophylactic treatment results in prevention and control ofbleeding, sustained control of bleeding, sustained protection frombleeding, and/or sustained benefit. Prophylaxis, e.g., sustainedprotection can be demonstrated by an increased AUC to last measured timepoint (AUC-LAST) and reduced clearance, resulting in increased terminalt1/2 compared to short acting FVIII. Prophylaxis can be demonstrated bybetter C_(max), better T_(max), and/or greater mean residence timeversus short-acting FVIII. In some embodiments, prophylaxis results inno spontaneous bleeding episodes within about 24, 36, 48, 72, or 96hours (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93,94, 95, or 96 hours), after injection (e.g., the last injection). Incertain embodiments, prophylaxis results in greater than 30% (e.g.,greater than 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 96, 87, 88, 89, or 90%, for example, greater than 50%),mean reduction in annualized bleeding episodes with once weekly dosing(e.g., at 65 IU/kg).

“Subject,” as used herein means a human individual. Subject can be apatient who is currently suffering from a bleeding disorder or isexpected to be in need of such a treatment. In some embodiments, thesubject has never been previously treated with the clotting factor(i.e., the subject is a previously untreated subject or previouslyuntreated patient). In some embodiments, the subject is a fetus and themethods comprises administering the chimeric polypeptide to the motherof the fetus and the administration to the subject occurs from themother across the placenta. In some embodiments, the subject is a childor an adult. In some embodiments, the subject is a child less thanone-year-old, less than two-year-old, less than three-year-old, lessthan four-year-old, less than five-year-old, less than six-year-old,less than seven-year-old, less than eight-year-old, less thannine-year-old, less than ten-year-old, less than eleven-year-old, orless than twelve-year-old. In some embodiments, the child is less thanone-year old. In some embodiments, the child or adult subject develops ableeding disorder, wherein the onset of the symptoms of the bleedingdisorder is after the one-year-old age. In some embodiments, theadministration of the chimeric polypeptide to the subject is sufficientto prevent, inhibit, or reduce development of an immune responseselected from a humoral immune response, a cell-mediated immuneresponse, or both a humoral immune response and a cell-mediated immuneresponse against the clotting factor.

In some embodiments described herein, the subject is at risk ofdeveloping an inhibitory FVIII immune response. A subject can be at riskof developing an inhibitory FVIII immune response because, for example,the subject previously developed an inhibitory FVIII immune response orcurrently has an inhibitory FVIII immune response. In some embodiments,the subject developed an inhibitory FVIII immune response aftertreatment with a plasma-derived FVIII product. In some embodiments, thesubject developed an inhibitory FVIII immune response after treatmentwith a recombinant FVIII product. In some embodiments, the subjectdeveloped an inhibitory FVIII immune response after treatment with afull-length FVIII protein. In some embodiments, the subject developed aninhibitory FVIII immune response after treatment with FVIII proteincontaining a deletion, e.g., a full or partial deletion of the B domain.

In some embodiments, the subject developed an inhibitory FVIII immuneresponse after treatment with a FVIII product selected from the groupconsisting of ADVATE®, RECOMBINATE®, KOGENATE FS®, HELIXATE FS®,XYNTHA®/REFACTO AB®, HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®,ALPHANATE®, KOATE-DVI®, AND HYATE:C®.

In some embodiments, the immune response after treatment with a clottingfactor, e.g., FVIII, comprises production of inhibitory antibodies tothe clotting factor. In some embodiments, the inhibitory antibodyconcentration is at least 0.6 Bethesda Units (BU). In some embodiments,the inhibitory antibody concentration is at least 5 BU. In someembodiments, the inhibitory antibody concentration is at least 0.5, atleast 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, atleast 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, atleast 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0BU.

In some embodiments, the immune response after treatment with a clottingfactor, e.g., FVIII, comprises a cell-mediated immune response. In someembodiments, the immune response comprises a cell-mediated immuneresponse. The cell-mediated immune response can comprise that release ofa cytokine selected from the group consisting of IL-12, IL-4 and TNF-α.

In some embodiments, the immune response after treatment with a clottingfactor, e.g., FVIII, comprises a clinical symptom selected from thegroup consisting of: increased bleeding tendency, high clotting factorconsumption, lack of response to clotting factor therapy, decreaseefficacy of clotting factor therapy, and shortened half-life of clottingfactor. In some embodiments, the subject has a mutation or deletion inthe clotting factor gene. In some embodiments, the subject has arearrangement in the clotting factor gene. In some embodiments, thesubject has severe hemophilia. In some embodiments, the subject has arelative who has previously developed an inhibitory immune responseagainst the clotting factor. In some embodiments, the subject isreceiving interferon therapy. In some embodiments, the subject isreceiving antiviral therapy.

In some embodiments, the subject at risk has previously developed aninhibitory immune response to a therapeutic protein other than FVIII.

In addition, the subject can also be at risk of developing an inhibitoryFVIII immune response as a result of a number of environmental orgenetic factors that increase the likelihood the subject will develop aninhibitory immune response. Factors that increase the likelihood that asubject will develop an inhibitory immune response are known. See e.g.,Kasper, C. “Diagnosis and Management of Inhibitors to Factors VIII andIX—An Introductory Discussion for Physicians,” Treatment of Hemophilia34 (2004), which is herein incorporated by reference in its entirety.For example, inhibitors arise more commonly in severe hemophilia (e.g.,FVIII baseline level of less than 1%) than in mild or moderatehemophilia. See Report of Expert Meeting on FVIII Products and InhibitorDevelopment, European Medicines Agency (Feb. 28, 2006-Mar. 2, 2006),which is herein incorporated by reference in its entirety.

As a result of the fact that genetic factors can increase the likelihoodthat a subject will develop an inhibitory immune response, a subjectwith at least one family member (e.g., a sibling, parent, child,grandparent, aunt, uncle, or cousin) who has developed an inhibitoryimmune response to FVIII or another therapeutic protein can be at riskof developing an inhibitory FVIII immune response.

Inhibitors are common in patients with genetic mutations that preventexpression of FVIII such as large genetic deletions, nonsense mutationscausing premature stop codons, and large inversions in the FVIII gene.Thus, in some embodiments, a subject at risk of developing an inhibitoryFVIII immune response comprises a mutation, deletion, or rearrangementin a FVIII gene. In some embodiments, a subject at risk of developing aninhibitory FVIII immune response comprises a mutation in FVIII thatprevents FVIII from binding to T cells. An association of inhibitorswith large rearrangements of FVIII genes has been observed. SeeAstermark et al., Blood 107:3167-3172 (2006), which is hereinincorporated by reference in its entirety. Thus, in some embodiments, aFVIII-Fc fusion protein is administered to a subject with a largerearrangement in a FVIII gene, for example, an intron 22 inversion or anintron 1 inversion. In some embodiments, FVIII-Fc is administered to asubject with two large rearrangements in FVIII. An association ofinhibitors with null mutations has also been observed. Astermark et al.,Blood 108:3739-3745 (2006). Thus, in some embodiments, a FVIII-Fc fusionprotein is administered to a subject with a null mutation.

Inhibitors also arise more commonly after treatment with exogenousclotting factors on only a few occasions. Thus, in some embodiments, asubject at risk of developing an inhibitory immune response has had lessthan 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, or 5 exposuredays. In some embodiments, a subject at risk of developing an inhibitoryimmune response has not been previously exposed to treatment with FVIII.

In some embodiments, the subject is a fetus. Immune tolerance can beinduced in a fetus by administering a chimeric polypeptide comprising aFVIII portion and an Fc portion to the mother of the fetus.

Inhibitors also arise more commonly in black African descent. See e.g.,Kasper, C. “Diagnosis and Management of Inhibitors to Factors VIII andIX—An Introductory Discussion for Physicians,” Treatment of Hemophilia34 (2004), which is herein incorporated by reference in its entirety.Thus, in some embodiments, a subject at risk of developing an inhibitoryimmune response is of black African descent.

Genetic mutations in genes other than FVIII have also been linked withan increased risk of developing an inhibitory immune response. Forexample, the TNF-α-308G>A polymorphism within Hap2, which is associatedwith increased constitutive and inducible transcription levels of TNFhas been linked with an increased risk of developing an inhibitoryimmune response. See Astermark et al., Blood 108: 3739-3745 (2006),which is herein incorporated by reference in its entirety. Thus, in someembodiments, a subject at risk of developing an inhibitory immuneresponse has a genetic polymorphism associated with increased TNF-α. Insome embodiments, the polymorphism is the TNF-α-308G>A polymorphism. Insome embodiments, a subject at risk of developing an inhibitory immuneresponse has a polymorphism in an IL10 gene, e.g. a polymorphismassociated with increased secretion of IL10. In some embodiments,FVIII-Fc is administered to a subject with the allele 134 of the IL10Gmicrosatellite in the promote region of the IL10 gene. See Astermark etal. Hemostatis, Thrombosis, and Vascular Biology 108: 3739-3745 (2006),which is herein incorporated by reference in its entirety.

In some embodiments, a subject at risk of developing an inhibitoryimmune response has a genetic polymorphism associated with decreasedCTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) expression. In someembodiments, a subject at risk of developing an inhibitory immuneresponse has a mutation in DR15 (HLA-DR15) or DQB0602 MHC (Majorhistocompatibility complex) Class II molecules. Other MHC Class IImolecules associated with the development of an inhibitory immuneresponse in subjects with hemophilia are A3, B7, C7, DQA0102, C2,DQA0103, DQB0603, and DR13 (see Inhibitors in Patients with Hemophilia,E. C. Rodriguez-Merchan & C. A. Lee, Eds., Blackwell Science, Ltd,2002).

In some embodiments, the subject at risk for developing an inhibitoryimmune response has not been previously exposed to clotting factor,e.g., FVIII. In some embodiments, the subject at risk for developing aninhibitory immune response to a clotting factor, e.g., FVIII, has beenexposed to FVIII. In some embodiments, the subject at risk fordeveloping an inhibitory immune response to a clotting factor, e.g.,FVIII, has had less than 150, less than 50, or less than 20 FVIIIexposure days. In some embodiments, the subject has had at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 daysof clotting factor exposure. In some embodiments, the subject has had atleast 25, 30, 35, 40, 45, or 50 days of clotting factor exposure. Insome embodiments, the subject has had at least 60, 70, 80, 90, 100, 110,120, 130, 140 or 150 days of clotting factor exposure.

In some embodiments, the inhibitory immune response is an inhibitoryFVIII immune response. In some embodiments, the inhibitory FVIII immuneresponse developed in response to a FVIII product selected from thegroup consisting of: ADVATE®, RECOMBINATE®, KOGENATE FS®, HELIXATE FS®,XYNTHA®/REFACTO ABC), HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®,ALPHANATE®, KOATE-DVI®, AND HYATE:C®. In some embodiments, theinhibitory immune response is an inhibitory FVIII immune responsedeveloped in response to a recombinant FVIII product.

In some embodiments, a subject at risk of developing an inhibitoryimmune response is receiving or has recently received animmunostimulatory therapy. For example, inhibitors have also beenreported in HCV positive hemophilia A patients undergoing treatment withinterferon as well as in HIV positive hemophilia A patients having animmune reconstitution inflammatory syndrome associated withanti-retroviral therapy. See Report of Expert Meeting on FVIII Productsand Inhibitor Development, European Medicines Agency (Feb. 28, 2006-Mar.2, 2006), which is herein incorporated by reference in its entirety.Thus, in some embodiments, a subject at risk of developing an inhibitoryimmune response is receiving interferon therapy. In some embodiments, asubject at risk of developing an inhibitory immune response is receivingan anti-retroviral therapy and having an immune reconstitutioninflammatory syndrome.

An inhibitory FVIII immune response can be determined based on clinicalresponses to FVIII treatment. Clinical presentations of FVIII inhibitorsare known and described, for example, in Kasper, C. “Diagnosis andManagement of Inhibitors to Factors VIII and IX—An IntroductoryDiscussion for Physicians,” Treatment of Hemophilia 34 (2004), which isherein incorporated by reference in its entirety. For example, thepresence of an inhibitor makes controlling hemorrhages for difficult, soimmune responses to FVIII are signaled by an increased bleedingtendency, high FVIII consumption, lack of response to FVIII therapy,decreased efficacy of FVIII therapy, and/or shortened half-life ofFVIII.

Inhibitory FVIIII immune responses can also be determined usinglaboratory tests such as the Bethesda test or the Nijmegan modificationof the Bethesda test. A level of at least 0.6 Bethesda Units (BU) canindicate the presence of an inhibitory immune response. A level of atleast 5 BU can indicate the presence of a high titer inhibitor.Measurements of the in vivo recovery and half-life of bolus FVIIIinfusion can also be used.

In some embodiments, a subject at risk of developing an inhibitoryimmune response has previously had an inhibitory immune response with apeak titer of at least 0.5, at least 0.6, at least 0.7, at least 0.8, atleast 0.9, at least 1.0, at least 1.5, at least 2.0, at least 3.0, atleast 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, atleast 9.0, or at least 10.0 BU.

In some embodiments provided herein, the methods comprise determining ifa subject is at risk of developing an inhibitory FVIII immune responseand administering to the subject a chimeric polypeptide comprising aFVIII portion and an Fc portion if the subject is at risk. Thus, in someembodiments, the methods comprise determining if the subject has amutation, deletion, or rearrangement in FVIII and administering achimeric polypeptide if the subject does. In some embodiments, themethods comprise determining if the subject produces FVIII protein andadministering a chimeric polypeptide if the subject does not. In someembodiments, the methods comprise determining if the subject has mild,moderate, or severe hemophilia, and administering a chimeric polypeptideif the subject has severe hemophilia. In some embodiments, the methodscomprise determining if the subject has an inhibitory FVIII immuneresponse, e.g., by assessing clinical manifestations of an immuneresponse, measuring anti-FVIII antibody levels, titers, or activities,or measuring cell-mediated immune response (e.g., by measuring levels ofcytokines) and administering a chimeric polypeptide if the subject hasat least one of these indicators.

“Therapeutic dose,” as used herein, means a dose that achieves atherapeutic goal, as described herein. The calculation of the requireddosage of FVIII is based upon the empirical finding that, on average, 1IU of FVIII per kg body weight raises the plasma FVIII activity byapproximately 2 IU/dL. The required dosage is determined using thefollowing formula:Required units=body weight (kg)×desired FVIII rise (IU/dL or % ofnormal)×0.5 (IU/kg per IU/dL)

The therapeutic doses that can be used in the methods of the presentdisclosure are about 10-100 IU/kg, more specifically, 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg, and morespecifically, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 IU/kg.

Additional therapeutic doses that can be used in the methods of thepresent disclosure are about 10 to about 150 IU/kg, more specifically,about 100-110, 110-120, 120-130, 130-140, 140-150 IU/kg, and morespecifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150IU/kg.

“Variant,” as used herein, refers to a polynucleotide or polypeptidediffering from the original polynucleotide or polypeptide, but retainingessential properties thereof, e.g., FVIII coagulant activity or Fc (FcRnbinding) activity. Generally, variants are overall closely similar, and,in many regions, identical to the original polynucleotide orpolypeptide. Variants include, e.g., polypeptide and polynucleotidefragments, deletions, insertions, and modified versions of originalpolypeptides.

Variant polynucleotides can comprise, or alternatively consist of, anucleotide sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or99% identical to, for example, the nucleotide coding sequence in SEQ IDNO:1, 3, or 5 (the FVIII portion, the Fc portion, individually ortogether) or the complementary strand thereto, the nucleotide codingsequence of known mutant and recombinant FVIII or Fc such as thosedisclosed in the publications and patents cited herein or thecomplementary strand thereto, a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2, 4, or 6 (the FVIII portion, the Fc portion,individually or together), and/or polynucleotide fragments of any ofthese nucleic acid molecules (e.g., those fragments described herein).Polynucleotides which hybridize to these nucleic acid molecules understringent hybridization conditions or lower stringency conditions arealso included as variants, as are polypeptides encoded by thesepolynucleotides as long as they are functional.

Variant polypeptides can comprise, or alternatively consist of, an aminoacid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to, for example, the polypeptide sequence shown in SEQ IDNOS:2, 4, or 6 (the FVIII portion, the Fc portion, individually ortogether), and/or polypeptide fragments of any of these polypeptides(e.g., those fragments described herein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence, it is intended thatthe nucleotide sequence of the nucleic acid is identical to thereference sequence except that the nucleotide sequence can include up tofive point mutations per each 100 nucleotides of the referencenucleotide sequence. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence can bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencecan be inserted into the reference sequence. The query sequence can be,for example, the entire sequence shown in SEQ ID NO:1 or 3, the ORF(open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toa nucleotide sequence or polypeptide of the present disclosure can bedetermined conventionally using known computer programs. In oneembodiment, a method for determining the best overall match between aquery sequence (reference or original sequence) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.,Comp. App. Biosci. 6:237-245 (1990), which is herein incorporated byreference in its entirety In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. In another embodiment, parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present disclosure. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made for thepurposes of the present disclosure.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the presentdisclosure, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence can include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence can be inserted, deleted, (indels) orsubstituted with another amino acid. These alterations of the referencesequence can occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences of SEQ ID NO:2 (the FVIII portion, the Fc portion,individually or together) or 4, or a known FVIII or Fc polypeptidesequence, can be determined conventionally using known computerprograms. In one embodiment, a method for determining the best overallmatch between a query sequence (reference or original sequence) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990), incorporatedherein by reference in its entirety. In a sequence alignment the queryand subject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment is inpercent identity. In another embodiment, parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentdisclosure. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made for thepurposes of the present disclosure.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In one embodiment, thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In another embodiment,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. In other embodiments, variants in which5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in anycombination. Polynucleotide variants can be produced for a variety ofreasons, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to others, e.g., a bacterial host suchas E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentdisclosure. Alternatively, non-naturally occurring variants can beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants can be generated to improve or alter thecharacteristics of the polypeptides. For instance, one or more aminoacids can be deleted from the N-terminus or C-terminus of the secretedprotein without substantial loss of biological function. Ron et al., J.Biol. Chem. 268: 2984-2988 (1993), incorporated herein by reference inits entirety, reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988), incorporated herein by reference in its entirety.)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993),incorporated herein by reference in its entirety) conducted extensivemutational analysis of human cytokine IL-1a. They used randommutagenesis to generate over 3,500 individual IL-1a mutants thataveraged 2.5 amino acid changes per variant over the entire length ofthe molecule. Multiple mutations were examined at every possible aminoacid position. The investigators found that “[m]ost of the moleculecould be altered with little effect on either [binding or biologicalactivity].” (See Abstract.) In fact, only 23 unique amino acidsequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

As stated above, polypeptide variants include, e.g., modifiedpolypeptides. Modifications include, e.g., acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporatedherein by reference in its entirety), proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. In some embodiments, FVIII ismodified, e.g., pegylated, at any convenient location. In someembodiments, FVIII is pegylated at a surface exposed amino acid ofFVIII, e.g., a surface exposed cysteine, which can be an engineeredcysteine. Id. In some embodiments, modified FVIII, e.g., pegylatedFVIII, is a long-acting FVIII.

“Volume of distribution at steady state (Vss),” as used herein, has thesame meaning as the term used in pharmacology, which is the apparentspace (volume) into which a drug distributes. Vss=the amount of drug inthe body divided by the plasma concentration at steady state.

“About,” as used herein for a range, modifies both ends of the range.Thus, “about 10-20” means “about 10 to about 20.”

The chimeric polypeptide used herein can comprise processed FVIII orsingle chain FVIII or a combination thereof “Processed FVIII,” as usedherein means FVIII that has been cleaved at Arginine 1648 (forfull-length FVIII) or Arginine 754 (for B-domain deleted FVIII), i.e.,intracellular processing site. Due to the cleavage at the intracellularprocessing site, processed FVIII comprises two polypeptide chains, thefirst chain being a heavy chain and the second chain being a lightchain. For example, the processed FVIII-Fc fusion protein (i.e., Heavychain and Light chain fused to Fc) run at approximately 90 kDa and 130kDa on a non-reducing SDS-PAGE, respectively, and 90 kDa and 105 kDa ona reducing SDS-PAGE, respectively. Therefore, in one embodiment, atleast about 50%, about 60%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% of the FVIII portion in the chimeric polypeptide is processedFVIII.

In another embodiment, about 50%, about 60%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% of the FVIII portion in the chimericpolypeptide is processed FVIII. In a particular embodiment, the chimericpolypeptide comprising processed FVIII is purified (or isolated) fromthe chimeric polypeptide comprising single chain FVIII, and at leastabout 90%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% of the FVIII portion in the chimeric polypeptide is processedFVIII.

The terms “Single chain FVIII” or “SC FVIII” as used herein mean FVIIIthat has not been cleaved at the Arginine site (residue 1648 forfull-length FVIII (i.e., residue 1667 of SEQ ID NO: 6) or residue 754for B-domain deleted FVIII (i.e., residue 773 of SEQ ID NO: 2).Therefore, single chain FVIII in the chimeric polypeptide used hereincomprises a single chain. In one embodiment, the single chain FVIIIcontains an intact intracellular processing site. The single chainFVIII-Fc fusion protein can run at approximately 220 kDa on a nonreducing SDS-PAGE and at approximately 195 kDa on a reducing SDS-PAGE.

In one embodiment, the chimeric polypeptide comprising single chainFVIII is purified (or isolated) from the chimeric polypeptide comprisingprocessed FVIII, and at least about 30%, about 40%, about 50%, about60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 99%, or about 100% of the FVIII portion of the chimericpolypeptide used herein is single chain FVIII. In another embodiment, atleast about 1%, about 5%, about 10%, about 15%, about 20%, or about 25%of the FVIII portion of the chimeric polypeptide is single chain FVIII.In other embodiments, about 1%-about 10%, about 5%-about 15%, about10%-about 20%, about 15%-about 25%, about 20%-about 30%, about 25%-about35%, about 30%-about 40% of the FVIII portion of the chimericpolypeptide used herein is single chain FVIII.

In a particular embodiment, about 1%, about 5%, about 10%, about 15%,about 20% or about 25% of the FVIII portion of the chimeric polypeptideused herein is single chain FVIII. In other embodiments, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% of theFVIII portion of the chimeric polypeptide used herein is single chainFVIII. In some embodiments, the ratio of the single chain FVIII to theprocessed FVIII of the chimeric polypeptide is (a) about 25% of singlechain FVIII and about 75% of processed FVIII; (b) about 20% of singlechain FVIII and about 80% of processed FVIII; (c) about 15% of singlechain FVIII and about 85% of processed FVIII; (d) about 10% of singlechain FVIII and about 90% of processed FVIII; (e) about 5% of singlechain FVIII and about 95% of processed FVIII; (f) about 1% of singlechain FVIII and about 99% of processed FVIII; or (g) about 100% ofprocessed FVIII.

In other embodiments, the ratio of the single chain FVIII to theprocessed FVIII of the chimeric polypeptide is (a) about 30% of singlechain FVIII and about 70% of processed FVIII; (b) about 40% of singlechain FVIII and about 60% of processed FVIII; (c) about 50% of singlechain FVIII and about 50% of processed FVIII; (d) about 60% of singlechain FVIII and about 40% of processed FVIII; (e) about 70% of singlechain FVIII and about 30% of processed FVIII; (f) about 80% of singlechain FVIII and about 20% of processed FVIII; (g) about 90% of singlechain FVIII and about 10% of processed FVIII; (h) about 95% of singlechain FVIII and about 5% of processed FVIII; (i) about 99% of singlechain FVIII and about 1% of processed FVIII; or (j) about 100% of singlechain FVIII.

The FVIII portion in the chimeric polypeptide used herein has FVIIIactivity. FVIII activity can be measured by any known methods in theart. For example, one of those methods can be a chromogenic assay. Thechromogenic assay mechanism is based on the principles of the bloodcoagulation cascade, where activated FVIII accelerates the conversion ofFactor X into Factor Xa in the presence of activated Factor IX,phospholipids and calcium ions. The Factor Xa activity is assessed byhydrolysis of a p-nitroanilide (pNA) substrate specific to Factor Xa.The initial rate of release of p-nitroaniline measured at 405 nM isdirectly proportional to the Factor Xa activity and thus to the FVIIIactivity in the sample.

The chromogenic assay is recommended by the FVIII and Factor IXSubcommittee of the Scientific and Standardization Committee (SSC) ofthe International Society on Thrombosis and Hemostatsis (ISTH). Since1994, the chromogenic assay has also been the reference method of theEuropean Pharmacopoeia for the assignment of FVIII concentrate potency.Thus, in one embodiment, the chimeric polypeptide comprising singlechain FVIII has FVIII activity comparable to a chimeric polypeptidecomprising processed FVIII (e.g., a chimeric polypeptide consistingessentially of or consisting of two Fc portions and processed FVIII,wherein said processed FVIII is fused to one of the two Fc portions),when the FVIII activity is measured in vitro by a chromogenic assay.

In another embodiment, the chimeric polypeptide comprising single chainFVIII of this disclosure has a Factor Xa generation rate comparable to achimeric polypeptide comprising processed FVIII (e.g., a chimericpolypeptide consisting essentially of or consisting of two Fc portionsand processed FVIII, wherein the processed FVIII is fused to one Fc ofthe two Fc portions).

In order to activate Factor X to Factor Xa, activated Factor IX (FactorIXa) hydrolyzes one arginine-isoleucine bond in Factor X to form FactorXa in the presence of Ca²⁺, membrane phospholipids, and a FVIIIcofactor. Therefore, the interaction of FVIII with Factor IX is criticalin coagulation pathway. In certain embodiments, the chimeric polypeptidecomprising single chain FVIII can interact with Factor IXa at a ratecomparable to a chimeric polypeptide comprising processed FVIII (e.g., achimeric polypeptide consisting essentially of or consisting of two Fcportions and processed FVIII, wherein the processed FVIII is fused toone Fc of the two Fc portions).

In addition, FVIII is bound to von Willebrand Factor while inactive incirculation. FVIII degrades rapidly when not bound to vWF and isreleased from vWF by the action of thrombin. In some embodiments, thechimeric polypeptide comprising single chain FVIII binds to vonWillebrand Factor at a level comparable to a chimeric polypeptidecomprising processed FVIII (e.g., a chimeric polypeptide consistingessentially of or consisting of two Fc portions and processed FVIII,wherein the processed FVIII is fused to one Fc of the two Fc portions).

FVIII can be inactivated by activated protein C in the presence ofcalcium and phospholipids. Activated protein C cleaves FVIII heavy chainafter Arginine 336 in the A1 domain, which disrupts a Factor X substrateinteraction site, and cleaves after Arginine 562 in the A2 domain, whichenhances the dissociation of the A2 domain as well as disrupts aninteraction site with the Factor IXa. This cleavage also bisects the A2domain (43 kDa) and generates A2-N (18 kDa) and A2-C (25 kDa) domains.Thus, activated protein C can catalyze multiple cleavage sites in theheavy chain. In one embodiment, the chimeric polypeptide comprisingsingle chain FVIII is inactivated by activated Protein C at a levelcomparable to a chimeric polypeptide comprising processed FVIII (e.g., achimeric polypeptide consisting essentially of or consisting of two Fcportions and processed FVIII, wherein the processed FVIII is fused toone Fc of the two Fc portions).

In other embodiments, the chimeric polypeptide comprising single chainFVIII has FVIII activity in vivo comparable to a chimeric polypeptidecomprising processed FVIII (e.g., a chimeric polypeptide consistingessentially of or consisting of two Fc portions and processed FVIII,wherein the processed FVIII is fused to one Fc of the two Fc portions).In a particular embodiment, the chimeric polypeptide comprising singlechain FVIII is capable of protecting a HemA mouse at a level comparableto a chimeric polypeptide comprising processed FVIII (e.g., a chimericpolypeptide consisting essentially of or consisting of two Fc portionsand processed FVIII, wherein said processed FVIII is fused to one Fc ofthe two Fc portions) in a HemA mouse tail vein transection model.

The term “comparable” as used herein means a compared rate or levelresulted from using the chimeric polypeptide is equal to, substantiallyequal to, or similar to the reference rate or level. The term “similar”as used herein means a compared rate or level has a difference of nomore than 10% or no more than 15% from the reference rate or level(e.g., FXa generation rate by a chimeric polypeptide consistingessentially of or consisting of two Fc portions and processed FVIII,wherein said processed FVIII is fused to one Fc of the two Fc portions).The term “substantially equal” means a compared rate or level has adifference of no more than 0.01%, 0.5% or 1% from the reference rate orlevel.

The present disclosure further includes a composition comprising achimeric polypeptide having FVIII activity, wherein at least about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about90%, about 95%, or about 99% of the chimeric polypeptide comprises aFVIII portion, which is single chain FVIII and a second portion. Inanother embodiment, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, or about 99% of the chimeric polypeptide in the compositionis single chain FVIII. In other embodiments, the second portion is anFc. In yet other embodiments, the chimeric polypeptide comprises anotherhalf-life extending moiety, e.g., albumin.

In still other embodiments, the composition of the present disclosurecomprises a combination of a chimeric polypeptide comprising processedFVIII and a chimeric polypeptide comprising single chain FVIII, (a)wherein about 30% of the FVIII portion of the chimeric polypeptide issingle chain FVIII, and about 70% of the FVIII portion of the chimericpolypeptide is processed FVIII; (b) wherein about 40% of the FVIIIportion of the chimeric polypeptide is single chain FVIII, and about 60%of the FVIII portion of the chimeric polypeptide is processed FVIII; (c)wherein about 50% of the FVIII portion of the chimeric polypeptide issingle chain FVIII, and about 50% of the FVIII portion of the chimericpolypeptide is processed FVIII; (d) wherein about 60% of the FVIIIportion of the chimeric polypeptide is single chain FVIII and about 40%of the FVIII portion of the chimeric polypeptide being processed FVIII;(e) wherein about 70% of the FVIII portion of the chimeric polypeptideis single chain FVIII and about 30% of the FVIII portion of the chimericpolypeptide is processed FVIII; (f) wherein about 80% of the FVIIIportion of the chimeric polypeptide is single chain FVIII and about 20%of the FVIII portion of the chimeric polypeptide is processed FVIII; (g)wherein about 90% of the FVIII portion of the chimeric polypeptide issingle chain FVIII and about 10% of the FVIII portion of the chimericpolypeptide is processed FVIII; (h) wherein about 95% of the FVIIIportion of the chimeric polypeptide is single chain FVIII and about 5%of the FVIII portion of the chimeric polypeptide is processed FVIII; (i)wherein about 99% of the FVIII portion of the chimeric polypeptide issingle chain FVIII and about 1% of the FVIII portion of the chimericpolypeptide is processed FVIII; or (j) wherein about 100% of the FVIIIportion of the chimeric polypeptide is single chain FVIII.

In certain embodiments, the composition of the present disclosure hasFVIII activity comparable to the composition comprising processed FVIII(e.g., a composition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed FVIII,wherein said processed FVIII is fused to one of the two Fc portions),when the FVIII activity is measured in vitro by a chromogenic assay.

In other embodiments, the composition of the disclosure has a Factor Xageneration rate comparable to a composition comprising processed FVIII(e.g., a composition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed FVIII,wherein the processed FVIII is fused to one Fc of the two Fc portions).In still other embodiments, the composition comprising single chainFVIII can interact with Factor IXa at a rate comparable to a compositioncomprising processed FVIII (e.g., a composition comprising a chimericpolypeptide, which consists essentially of or consists of two Fcportions and processed FVIII, wherein the processed FVIII is fused toone Fc).

In further embodiments, the single chain FVIII in the chimericpolypeptide of the present composition is inactivated by activatedProtein C at a level comparable to processed FVIII in a chimericpolypeptide of a composition (e.g., a composition comprising a chimericpolypeptide, which consists essentially of or consists of two Fcportions and processed FVIII, wherein the processed FVIII is fused toone Fc of the two Fc portions). In a particular embodiment, thecomposition comprising single chain FVIII has FVIII activity in vivocomparable to the composition comprising processed FVIII (e.g., acomposition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed FVIII,wherein the processed FVIII is fused to one Fc of the two Fc portions).In some embodiments, the composition comprising single chain FVIII ofthe present disclosure is capable of protecting HemA mouse at a levelcomparable to the composition comprising processed FVIII (e.g., acomposition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed FVIII,wherein said processed FVIII is fused to one Fc of the two Fc portions)in HemA mouse tail vein transection model.

The present disclosure further provides a method for treating a bleedingcondition in a human subject using the compositions disclosed herein. Anexemplary method comprises administering to the subject in need thereofa therapeutically effective amount of a pharmaceuticalcomposition/formulation comprising a chimeric polypeptide having FVIIIactivity, wherein at least about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% ofthe chimeric polypeptide comprises a FVIII portion, which is singlechain FVIII, and a second portion.

The bleeding condition can be caused by a blood coagulation disorder. Ablood coagulation disorder can also be referred to as a coagulopathy. Inone example, the blood coagulation disorder, which can be treated with apharmaceutical composition of the current disclosure, is hemophilia orvon Willebrand disease (vWD). In another example, the blood coagulationdisorder, which can be treated with a pharmaceutical composition of thepresent disclosure is hemophilia A.

In some embodiments, the type of bleeding associated with the bleedingcondition is selected from hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath.

In other embodiments, the subject suffering from bleeding condition isin need of treatment for surgery, including, e.g., surgical prophylaxisor peri-operative management. In one example, the surgery is selectedfrom minor surgery and major surgery. Exemplary surgical proceduresinclude tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranialsurgery, intra-abdominal surgery, intrathoracic surgery, jointreplacement surgery (e.g., total knee replacement, hip replacement, andthe like), heart surgery, and caesarean section.

In another example, the subject is concomitantly treated with FIX.Because the compounds of the present disclosure are capable ofactivating FIXa, they could be used to pre-activate the FIXa polypeptidebefore administration of the FIXa to the subject.

The methods disclosed herein can be practiced on a subject in need ofprophylactic treatment or on-demand treatment.

The pharmaceutical compositions comprising at least 30% of single chainFVIII can be formulated for any appropriate manner of administration,including, for example, topical (e.g., transdermal or ocular), oral,buccal, nasal, vaginal, rectal or parenteral administration.

The term parenteral as used herein includes subcutaneous, intradermal,intravascular (e.g., intravenous), intramuscular, spinal, intracranial,intrathecal, intraocular, periocular, intraorbital, intrasynovial andintraperitoneal injection, as well as any similar injection or infusiontechnique. The composition can be also for example a suspension,emulsion, sustained release formulation, cream, gel or powder. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

In one example, the pharmaceutical formulation is a liquid formulation,e.g., a buffered, isotonic, aqueous solution. In another example, thepharmaceutical composition has a pH that is physiologic, or close tophysiologic. In other examples, the aqueous formulation has aphysiologic or close to physiologic osmolarity and salinity. It cancontain sodium chloride and/or sodium acetate. In some examples, thecomposition of the present disclosure is lyophilized. In someembodiments, the pharmaceutical composition does not comprise an immunecell. In some embodiments, the pharmaceutical composition does notcomprise a cell.

The present disclosure also provides a kit comprising (a) apharmaceutical composition comprising a chimeric polypeptide whichcomprises a clotting factor portion and an Fc portion and apharmaceutically acceptable carrier, and (b) instructions to administerto the composition to a subject in need of immune tolerance to theclotting factor. In some embodiments, the chimeric polypeptide in thekit comprises a FVIII portion, a FVII portion, or a FIX portion. Inother embodiments, the chimeric polypeptide in the kit is a FVIIImonomer dimer hybrid, a FVII monomer dimer hybrid, or a FIX monomerdimer hybrid. In some embodiments, the instructions further include atleast one step to identify a subject in need of immune tolerance to theclotting factor. In some embodiments, the step to identify the subjectsin need of immune tolerance includes one or more from the groupconsisting of:

-   -   (i) identifying a subject having a mutation or deletion in the        clotting factor gene;    -   (ii) identifying a subject having a rearrangement in the        clotting factor gene;    -   (iii) identifying a subject having a relative who has previously        developed an inhibitory immune response against the clotting        factor;    -   (iv) identifying a subject receiving interferon therapy;    -   (v) identifying a subject receiving anti-viral therapy;    -   (vi) identifying a subject having a genetic mutation in a gene        other than the gene encoding the clotting factor which is linked        with an increased risk of developing an inhibitory immune        response; and    -   (vii) two or more combinations thereof.

In some embodiments, the genetic mutation in a gene other than the geneencoding the clotting factor comprises one or more mutations selectedfrom the group consisting of:

-   -   (i) a genetic polymorphism associated with increased TNF-α;    -   (ii) a genetic polymorphism associated with increased IL10;    -   (iii) a genetic polymorphism associated with decreased CTLA-4;    -   (iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and    -   (v) has two or more combinations thereof.

Optionally associated with the kit's container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

EMBODIMENTS

E1. A method of inducing immune tolerance to a clotting factor in asubject in need thereof comprising administering to the subject achimeric polypeptide, wherein the chimeric polypeptide comprises aclotting factor portion and an Fc portion.

E2. A method of preventing or inhibiting development of an inhibitor toa clotting factor comprising administering to a subject in need ofimmune tolerance to the clotting factor a chimeric polypeptide, whereinthe chimeric polypeptide comprises a clotting factor portion and an Fcportion.

E3. The method of embodiment E1 or E2, wherein the subject would developan inhibitory immune response against the clotting factor ifadministered an equivalent dose of a polypeptide consisting of theclotting factor.

E4. The method of any one of embodiments E1 to E3, wherein the subjecthas developed an inhibitory immune response against the clotting factor.

E5. The method of any one of embodiments E1 to E4, wherein the subjecthas never been previously treated with the clotting factor.

E6. The method of any one of embodiments E1 to E5, wherein the clottingfactor portion comprises Factor VIII, Factor IX, Factor VII, VonWillebrand Factor, or a fragment thereof.

E7. The method of any one of embodiments E1 to E5, where in the subjectis a fetus and the method further comprises administering the chimericpolypeptide to the mother of the fetus and the administration to thesubject occurs from the mother across the placenta.

E8. The method of embodiment E7, wherein the clotting factor moietycomprises Factor VIII, Factor IX, Factor VII, Von Willebrand Factor, ora fragment thereof.

E9. The method of any one of embodiments E1 to E6, wherein the subjectis a child or an adult.

E10. The method of embodiment E9, wherein the subject is a child lessthan one-year-old, less than two-year-old, less than three-year-old,less than four-year-old, less than five-year-old, less thansix-year-old, less than seven-year-old, less than eight-year-old, lessthan nine-year-old, less than ten-year-old, less than eleven-year-old,or less than twelve-year-old.

E11. The method of embodiment E10, wherein the child is less thanone-year old.

E12. The method of embodiment E11, wherein the child or adult develops ableeding disorder, wherein the onset of the symptoms of the bleedingdisorder is after the one-year-old age.

E13. The method of any one of embodiments E1 to E12 wherein theadministration is sufficient to prevent, inhibit, or reduce developmentof an immune response selected from a humoral immune response, acell-mediated immune response, or both a humoral immune response and acell-mediated immune response against the clotting factor.

E14. The method of any one of embodiments E1 to E13, wherein thecomposition is administered in repeated doses.

E15. The method of embodiment E14, wherein each of the repeated doses isseparated from another by at least about 12 hours, at least about 24hours, at least about two days, at least about three days, at leastabout four days, at least about five days, at least about six days, atleast about seven days, at least about eight days, at least about ninedays, at least about ten days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, or at least about15 days.

E16. The method of embodiment E14 or E15, wherein the repeated dosescomprise at least about two doses, at least about five doses, at leastabout 10 doses, at least about 20 doses, at least about 25 doses, atleast about 30 doses, at least about 35 doses, at least about 40 doses,at least about 45 doses, at least about 50 doses, at least about 55doses, at least about 60 doses, at least about 65 doses, or at leastabout 70 doses.

E17. The method of embodiment E16, wherein the repeated doses comprisefrom about two doses to about 100 doses, from about five doses to about80 doses, from about 10 doses to about 70 doses, from about 10 doses toabout 60 doses, from about 10 doses to about 50 doses, from about 15doses to about 40 doses, from about 15 doses to about 30 doses, fromabout 20 doses to about 30 doses, or from about 20 doses to about 40doses.

E18. The method of embodiment E16 or E17, wherein the repeated dosescomprise about two doses, about five doses, about 10 doses, about 15doses, about 20 doses, about 25 doses, about 30 doses, about 35 doses,about 40 doses, about 45 doses, about 50 doses, about 55 doses, about 60doses, about 65 does, about 70 doses, about 75 doses, about 80 doses,about 90 doses, or about 100 doses.

E19. The method of any one of embodiments E14 to E18, wherein thesubject is further administered, after the repeated doses, apharmaceutical composition comprising a clotting factor protein whichcomprises the clotting factor, but does not comprise an Fc portion.

E20. The method of embodiment E19, wherein the clotting factor proteinis full length or mature clotting factor.

E21. The method of embodiment E19, wherein the clotting factor proteincomprises one or more half-life extending moiety other than an Fcportion.

E22. The method of any one of embodiments E1 to E21, wherein theadministration treats one or more bleeding episodes.

E23. The method of any one of embodiments E1 to E22, wherein theadministration prevents one or more bleeding episodes.

E24. The method of any one of embodiments E1 to E23, wherein theadministration is an episodic treatment of one or more bleedingepisodes.

E25. The method of any one of embodiments E1 to E23, wherein the subjectis in need of surgical prophylaxis, peri-operative management, ortreatment for surgery.

E26. The method of embodiment E25, wherein the surgery is minor surgery,major surgery, tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, total knee replacement, craniotomy, osteosynthesis, traumasurgery, intracranial surgery, intra-abdominal surgery, intrathoracicsurgery, or joint replacement surgery.

E27. The method of any one of embodiments E13 to E26, wherein the immuneresponse comprises production of inhibitory antibodies to the clottingfactor.

E28. The method of embodiment E27, wherein the antibody concentration isat least 0.6 Bethesda Units (BU).

E29. The method of embodiment E28, wherein the antibody concentration isat least 5 BU.

E30. The method of any one of embodiments E13 to E26, wherein the immuneresponse comprises a cell-mediated immune response.

E31. The method of embodiment E30, wherein the cell-mediated immuneresponse comprises the release of a cytokine selected from the groupconsisting of IL-12, IL-4, and TNF-α.

E32. The method of any one of embodiments E13 to E31, wherein the immuneresponse comprises a clinical symptom selected from the group consistingof: increased bleeding tendency, high clotting factor consumption, lackof response to clotting factor therapy, decreased efficacy of clottingfactor therapy, and shortened half-life of clotting factor.

E33. The method of any one of embodiments E1 to E32, wherein the subjecthas a mutation or deletion in the clotting factor gene.

E34. The method of any one of embodiments E1 to E32, wherein the subjecthas a rearrangement in the clotting factor gene.

E35. The method of any one of embodiments E1 to E34, wherein the subjecthas severe hemophilia.

E36. The method of any one of embodiments E1 to E34, wherein the subjecthas a relative who has previously developed an inhibitory immuneresponse against the clotting factor.

E37. The method of any one of embodiments E1 to E36, wherein the subjectis receiving interferon therapy.

E38. The method of any one of embodiments E1 to E37, wherein the subjectis receiving anti-viral therapy.

E39. The method of any one of embodiments E1 to E38, wherein the subjecthas a genetic polymorphism associated with increased TNF-α.

E40. The method of embodiment E39, wherein the polymorphism is TNF-α308G>A.

E41. The method of any one of embodiments E1 to E40, wherein the subjecthas a genetic polymorphism associated with increased IL10.

E42. The method of embodiment E41, wherein the polymorphism is allele134 of the IL10G microsatellite.

E43. The method of any one of embodiments E1 to E42, wherein the subjecthas a genetic polymorphism associated with decreased CTLA-4 expression.

E44. The method of any one of embodiments E1 to E43, wherein the subjecthas a mutation in DR15 or DQB0602 MHC Class II molecules.

E45. The method of any one of embodiments E1 to E44, wherein the subjecthas had less than 150 clotting factor exposure days (ED).

E46. The method of embodiment E45, wherein the subject has had less than50 ED.

E47. The method of embodiment E46, wherein the subject has had less than20 ED.

E48. The method of any one of embodiment E3 to E47, wherein theinhibitory FVIII immune response developed in response to a full lengthor mature FVIII clotting factor.

E49. The method of embodiment E3-E48, wherein the inhibitory immuneresponse is an inhibitory FVIII response developed in response to arecombinant FVIII product.

E50. The method of any one of embodiments E1 to E49, wherein theadministration reduces the number of antibodies to FVIII in the subjectcompared to the number prior to administration.

E51. The method of any one of embodiments E1 to E50, wherein theadministration reduces the titer of antibodies to FVIII in the subjectcompared to the titer prior to administration.

E52. The method of any one of embodiments E1 to E51, wherein theadministration reduces the level of a cytokine in the subject comparedto the level prior to administration.

E53. The method of any one of embodiments E1 to E52, wherein theadministration reduces the number of antibodies to FVIII in the subjectcompared to the number in the subject after a previous treatment with apolypeptide consisting of a FVIII polypeptide.

E54. The method of any one of embodiments E1 to E53, wherein theadministration reduces the titer of antibodies to FVIII in the subjectcompared to the titer in the subject after a previous treatment with apolypeptide consisting of a FVIII polypeptide.

E55. The method of any one of embodiments E1 to E54, wherein theadministration reduces the level of a cytokine in the subject comparedto the level in the subject after a previous treatment with apolypeptide consisting of a FVIII polypeptide.

E56. The method of any one of embodiments E1 to E55, wherein theadministration reduces the number of anti-clotting factor antibodies inthe subject compared to the number that would result from administrationof polypeptide consisting of the clotting factor portion to the subject.

E57. The method of any one of embodiments E1 to E56, wherein theadministration reduces the titer of anti-clotting factor antibodies inthe subject compared to the titer that would result from administrationof polypeptide consisting of the clotting factor portion to the subject.

E58. The method of any one of embodiments E1 to E57, wherein theadministration reduces the level of a cytokine in the subject comparedto the level that would result from administration of polypeptideconsisting of the clotting factor portion to the subject.

E59. The method of embodiment E52, E55, or E58, wherein the cytokine isselected from the group consisting of IL-12, IL-4, and TNF-α.

E60. The method of any one of embodiments E1 to E59, which furthercomprises prior to administration of the chimeric polypeptide,identifying that the subject has one or more characteristics selectedfrom the group consisting of:

(a) has a mutation or deletion in the gene encoding the clotting factor;

(b) has a rearrangement in the gene encoding the clotting factor;

(c) has a relative who has previously developed an inhibitory immuneresponse against the clotting factor;

(d) is receiving interferon therapy;

(e) is receiving anti-viral therapy;

(f) has a genetic mutation in a gene other than the gene encoding theclotting factor which is linked with an increased risk of developing aninhibitory immune response; and

(g) two or more combinations thereof.

E61. The method of embodiment E60, wherein the genetic mutation in agene other than the gene encoding the clotting factor comprises one ormore mutations selected from the group consisting of:

(i) a genetic polymorphism associated with increased TNF-α;

(ii) a genetic polymorphism associated with increased IL10;

(iii) a genetic polymorphism associated with decreased CTLA-4;

(iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and

(v) has two or more combinations thereof.

E62. The method of embodiment E61, wherein the polymorphism associatedwith increased TNF-α is 308G>A.

E63. The method of embodiment E61, wherein the polymorphism associatedwith increased IL10 is allele 134 of the IL10G microsatellite.

E64. The method of any one of embodiments E6, and E8 to E62, wherein theFVIII portion comprises the FVIII A3 domain.

E65. The method of any one of embodiments E6, and E8 to E63, wherein theFVIII portion comprises human FVIII.

E66. The method of any one of embodiments E6, and E8 to E64, wherein theFVIII portion has a full or partial deletion of the B domain.

E67. The method of any one of embodiments E6, and E8 to E66, wherein theFVIII portion is at least 90% or 95% identical to a FVIII amino acidsequence shown in TABLE 2 without a signal sequence (amino acids 20 to1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6).

E68. The method of any one of embodiments E6, and E8 to E66, wherein theFVIII portion is identical to a FVIII amino acid sequence shown in TABLE2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2 oramino acids 20 to 2351 of SEQ ID NO:6).

E69. The method of any one of embodiments E6, and E8 to E66, wherein theFVIII portion is at least 90% or 95% identical to a FVIII amino acidsequence shown in TABLE 2 with a signal sequence (amino acids 1 to 1457of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6).

E70. The method of any one of embodiments E6, and E8 to E66, wherein theFVIII portion is identical to a FVIII amino acid sequence shown in TABLE2 with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or aminoacids 1 to 2351 of SEQ ID NO:6).

E71. The method of any one of embodiments E6, and E8 to E70, wherein theFVIII portion has coagulation activity.

E72. The method of any one of embodiments E1 to E71, wherein the Fcportion is identical to the Fc amino acid sequence shown in TABLE 2(amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 ofSEQ ID NO:6).

E73. The method of any one of embodiments E1 to E72, wherein thechimeric polypeptide is in the form of a hybrid comprising a secondpolypeptide in association with the chimeric polypeptide, wherein thesecond polypeptide consists essentially of or consists of the Fc portionor the FcRn binding partner.

E74. The method of any one of embodiments E1 to E73, wherein thechimeric polypeptide is administered at each dose of 10-100 IU/kg.

E75. The method of embodiment E74, wherein the dose is 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg.

E76. The method of embodiment E75, wherein the dose is 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg.

E77. The method of any one of embodiments E1 to E76, wherein the subjecthas a bleeding condition selected from the group consisting of ableeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath.

E78. The method of embodiment E77, wherein the bleeding coagulationdisorder is hemophilia A.

E79. The method of any one of embodiments E6, and E8 to E63, wherein theclotting factor portion comprises Factor IX.

E80. The method of embodiment E79, wherein the Factor IX portion is atleast 90%, 95%, or 100% identical to a FIX amino acid sequence shown inTABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ IDNO:2; amino acids 20 to 2351 of SEQ ID NO:6).

E81. The method of any one of embodiments E6, and E8 to E63, wherein thechimeric polypeptide is a monomer dimer hybrid comprising a first chaincomprising a FIX portion and the Fc portion or the FcRn binding partnerand a second chain consisting essentially of or consisting of a Fcportion.

E82. The method of any one of embodiments E6, and E8 to E63, wherein thechimeric polypeptide comprises a Factor VII portion.

E83. The method of embodiment E82, wherein the chimeric polypeptide is amonomer dimer hybrid comprising a first chain comprising a FVII portionand the Fc portion and a second chain consisting essentially of orconsisting of a Fc portion.

E84. The method of embodiment E82 or E83, wherein the FVII portion isinactive FVII, activated FVII, or activatable FVII.

E85. A kit comprising (a) a pharmaceutical composition comprising achimeric polypeptide which comprises a clotting factor portion and an Fcportion or an FcRn binding partner portion and a pharmaceuticallyacceptable carrier, and (b) instructions to administer to thecomposition to a subject in need of immune tolerance to the clottingfactor.

E86. The kit of embodiment E85, wherein the chimeric polypeptidecomprises a FVIII portion, a FVII portion, or a FIX portion.

E87. The kit of embodiment E86, wherein the chimeric polypeptide is aFVIII monomer dimer hybrid, a FVII monomer dimer hybrid, or a FIXmonomer dimer hybrid.

E88. The kit of any one of embodiments E85 to E87, wherein theinstructions further include at least one step to identify a subject inneed of immune tolerance to the clotting factor.

E89. The kit of embodiment E88, wherein the step to identify thesubjects in need of immune tolerance includes one or more from the groupconsisting of:

(a) identifying a subject having a mutation or deletion in the clottingfactor gene;

(b) identifying a subject having a rearrangement in the clotting factorgene;

(c) identifying a subject having a relative who has previously developedan inhibitory immune response against the clotting factor;

(d) identifying a subject receiving interferon therapy;

(e) identifying a subject receiving anti-viral therapy;

(f) identifying a subject having a genetic mutation in a gene other thanthe gene encoding the clotting factor which is linked with an increasedrisk of developing an inhibitory immune response; and

(g) two or more combinations thereof.

E90. The kit of claim E89, wherein the genetic mutation in a gene otherthan the gene encoding the clotting factor comprises one or moremutations selected from the group consisting of:

(i) a genetic polymorphism associated with increased TNF-α;

(ii) a genetic polymorphism associated with increased IL10;

(iii) a genetic polymorphism associated with decreased CTLA-4;

(iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and

(v) has two or more combinations thereof.

E91. The method of any one of embodiments E1 to E84, further comprisingmeasuring the level of an inhibitory immune response after theadministration.

E92. The method of embodiment E91, wherein further comparing the levelof the inhibitory immune response after the administration with thelevel of the inhibitory immune response before the administration.

E93. The method of embodiments E91 or E92, wherein the inhibitory immuneresponse is development of antibodies against FVIII.

E94. The method of E91 or E92, wherein the inhibitory immune response iscytokine secretion.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents and publicationsreferred to herein are expressly incorporated by reference.

EXAMPLES Example 1 Cloning, Expression and Purification of rFVIIIFc

All molecular biology procedures were performed following standardtechniques. The coding sequence of human FVIII (Genbank accession numberNM 000132), including its native signal sequence, was obtained byreverse transcription-polymerase chain reactions (RT-PCR) from humanliver polyA RNA. Due to the large size of FVIII, the coding sequence wasobtained in several sections from separate RT-PCR reactions, andassembled through a series of PCR reactions, restriction digests andligations into an intermediate cloning vector containing a B domaindeleted (BDD) FVIII coding region with a fusion of serine 743 (S743) toglutamine 1638 (Q1638), eliminating 2682 bp from the B domain of fulllength FVIII. The human IgG1 Fc sequence (e.g., GenBank accession numberY14735) was obtained by PCR from a leukocyte cDNA library, and the finalexpression cassette was made in such a way that the BDD FVIII sequencewas fused directly to the N-terminus of the Fc sequence (hinge, CH2 andCH3 domains, beginning at D221 of the IgG1 sequence, EU numbering) withno intervening linker. For expression of the Fc chain alone, the mouseIgκ (kappa) light chain signal sequence was created with syntheticoligonucleotides and added to the Fc coding sequence using PCR to enablesecretion of this protein product. The FVIIIFc and Fc chain codingsequences were cloned into a dual expression vector, pBudCE4.1(Invitrogen, Carlsbad, Calif.).

HEK 293H cells (Invitrogen, Carlsbad, Calif.) were transfected with thepSYN-FVIII-013 plasmid using Lipofectamine transfection reagent(Invitrogen, Carlsbad, Calif.)), and a stable cell line was selectedwith zeocin. Cells were grown in serum free suspension culture, andrFVIIIFc protein purified from clarified harvest media using a fourcolumn purification process, including a FVIII-specific affinitypurification step (McCue J. et al., J. Chromatogr. A., 1216(45): 7824-30(2009)), followed by a combination of anion exchange columns and ahydrophobic interaction column.

Example 2 Characterization of rFVIIIFc

(a) Biochemical Characterization

Processed recombinant FVIII-Fc (rFVIIIFc) was synthesized as twopolypeptide chains, one chain consisting of BDD-FVIII (S743-Q1638fusion, 1438 amino acids) fused to the Fc domain (hinge, CH2 and CH3domains) of IgG1 (226 amino acids, extending from D221 to G456, EUnumbering), for a total chain length of 1664 amino acids, the otherchain consisting of the same Fc region alone (226 amino acids). Thoughcells transfected with the FVIIIFc/Fc dual expression plasmid wereexpected to secrete three products (FVIIIFc dimer, FVIIIFc monomer, andFc dimer), only the FVIIIFc monomer and Fc dimer were detected inconditioned media. Purified FVIIIFc was analyzed by non-reducing andreducing SDS-PAGE analysis (FIGS. 2A and 2B). For the nonreducedSDS-PAGE, bands were found migrating at approximately 90 kDa and 130kDa, consistent with the predicted molecular weights of the FVIIIFcheavy chain (HC) and light chain-dimeric Fc fusion (LCFc2) (FIG. 2A,lane 3). A third band was also detected at approximately 220 kDa,consistent with the predicted molecular weight for single chain FVIIIFc(SC FVIIIFc; HC+LCFc2), in which the arginine residue at position 754(1648 with respect to the full length sequence) is not cleaved duringsecretion. For the reduced SDS-PAGE analysis, major bands were seenmigrating at approximately 25 kDa, 90 kDa, 105 kDa, and 195 kDa,consistent with the predicted molecular weights for the single chain Fc,HC, LCFc, and SC FVIIIFc (FIG. 2B, lane 3). Cotransfection with humanPC5, a member of the proprotein convertase subtlisin/kexin (PCSK) typeproteases, resulted in full processing of the rFVIIIFc product (FIGS.2A, 2B, lane 2).

Densitometry analysis of several batches of rFVIIIFc after SDS-PAGEindicated greater than 98% purity of the expected bands. Size exclusionchromatography (SEC) was also used to assess the degree of aggregationpresent, and all batches were found to have aggregate levels at 0.5% orless.

rFVIIIFc structure was further analyzed by thrombin cleavage, reduction,and analysis by LC/UV and LC/MS. The four FVIII fragments generated bythrombin (by cleavages at three arginine residues, at positions 372, 740and 795 (795 corresponds to 1689 with respect to the full length FVIIIsequence), can be detected by UV absorbance (FIG. 2C), corresponding tothe following segments of the protein: Fc (peak 1), light-chain-Fc (peak2); the A1 domain from the heavy chain (peak 3) and the A2 domain fromthe heavy chain (peak 4). The 14 amino acid B domain linker and ˜6 kDaa3-related peptides are not detected by UV absorbance due to their smallsize.

The rFVIIIFc polypeptide produced without cotransfected processingenzymes exhibited 15-25% single chain FVIIIFc (SC FVIIIFc), whichdiffers from processed rFVIIIFc by a single peptide bond between R754and E755 (R1648/E1649 with respect to the full length FVIII). Thisisoform was purified and characterized in all of the biochemical assaysdescribed above, and found to be comparable to rFVIIIFc as shown below.The activity of purified single chain FVIIIFc was found to be similar torFVIIIFc in a chromogenic assay as well as by the various functionalassays described below.

(b) Measurement of FVIII Activity by Chromogenic and One-Stage aPTTAssays

FVIII activity was measured by a FVIII chromogenic assay. The averagespecific activity from four separate batches of rFVIIIFc was found to be9762±449 IU/mg by the chromogenic assay, corresponding to 2148±99IU/nmol. FVIII activity of single chain FVIII:Fc was also measured bythe chromogenic assay and compared to the completely processed rFVIIIFcor rFVIIIFc DS (containing about 25% single chain rFVIIIFc). As TABLE 3Ashows, single chain rFVIIIFc showed no significant difference in FVIIIactivity compared to the FVIII activity of completely processed FVIIIFcor rFVIIIFc DS by the chromogenic assay, both in the presence and theabsence of von Willebrand Factor (VWF). TABLE 3B shows that fullactivity of SCrFVIIIFc, as measured by one-stage activated partialthromboplastin time (aPTT) assay, was observed in the absence of VWF.

TABLE 3A FVIII Activity by Chromogenic Assay Chromogenic Specific MatrixSample Activity (IU/mg) % CV* FVIII rFVIIIFcDS (25% NP) 9066 2.49depleted (RECD-19189-09-013) plasma Single chain rFVIIIFc 8194 2.72(purified from RECD 19189-09-013) Completely Processed 9577 8.34rFVIIIFc (purified from an engineered cell line) FVIII rFVIIIFcDS (25%NP) 10801 8.92 and vWF (RECD-19189-09-013) depleted Single chainrFVIIIFc 9498 4.70 plasma (purified from RECD 19189-09-013) CompletelyProcessed 9569 4.54 rFVIIIFc (purified from an engineered cell line) *CV= coefficient of variation

TABLE 3B FVIII Activity by aPTT assay Coagulation (aPTT) SpecificActivity Matrix Sample (IU/mg) % CV FVIII- rFVIIIFcDS (25% NP) 8210 5.88depleted (RECD-19189-09-013) plasma Single chain rFVIIIFc 3108 6.57(purified from RECD 19189-09-013) Completely Processed 8683 3.57rFVIIIFc (purified from an engineered cell line) FVIII rFVIIIFcDS (25%NP) 15621 6.47 and vWF (RECD-19189-09-013) depleted Single chainrFVIIIFc 13572 2.41 plasma (purified from RECD 19189-09-013) CompletelyProcessed 15170 10.42 rFVIIIFc (purified from an engineered cell line)

(c) Activity in Xase Complex

FVIII activity was also measured in the context of the Xase complex, byincubating activated FIX and thrombin-activated REFACTO® or rFVIIIFcprotein on a phospholipid surface in the presence of calcium, andmonitoring the conversion of FX to FXa as measured by cleavage of achromogenic or fluorogenic substrate, from which FXa generation rateswere determined. This assay was then modified by varying one componentof the assay while keeping the others constant in order to examine theinteractions with each individual component.

The FXa generation rate was determined as a function of varyingphospholipid concentrations for rFVIIIFc DS, rBDD FVIII, and singlechain rFVIIIFc (FIG. 3A), using synthetic phospholipid vesicles (25%phosphotadyl serine/75% phosphotadyl choline). Both proteins were foundto have a similar activity profile, with peak activity at approximately156 μM phospholipids.

The FXa generation rate was then determined as a function of varying FXconcentrations, and K_(m) and V_(max) values calculated (FIG. 3B). Theactivity profiles for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFcwere found to be similar, with similar K_(m) and V_(max) (TABLE 4).Finally, the FXa generation rate was determined as a function of varyingFIX concentrations (FIG. 3C). The activity profiles appeared similar,with similar K_(d) and V_(max) (TABLE 5). Similar results were obtainedusing platelets as a phospholipid source (unpublished data, June 2009).

TABLE 4 FXa Generation Parameters for FVIII Proteins on PhospholipidsLipid Source Molecule Km (nM) Vmax (nM/min) 25% PS-75% PC rFVIIIFc DS55.0 ± 5.9 65.6 ± 8.6  rBDD FVIII 51.0 ± 8.7 73.5 ± 10.1 NP rFVIIIFc53.2 ± 7.5 56.0 ± 13.8

TABLE 5 FIXa Interactions with FVIII Proteins Lipid Source Molecule Km(nM) Vmax (nM/min) 25% PS-75% PC rFVIIIFc DS 2.8 ± 0.4 4.5 ± 0.3 rBDDFVIII 2.5 ± 0.3 4.0 ± 1.0 NP rFVIIIFc 2.3 ± 0.2 3.8 ± 0.4

(d) Inactivation by APC

Once active, FVIII is inactivated by cleavage by activated protein C(APC), as well as by dissociation of the A2 domain. rFVIIIFc and rBDDFVIII were both activated by thrombin, then incubated with APC fordifferent times and activity determined in a FXa generation assay (FIG.4). In the absence of thrombin activation, little FXa generation wasdetected, and this was increased significantly with thrombin digestion.Treatment with APC for 90 minute led to a significant decrease in FXageneration rates, similar to non-activated samples, and these resultswere similar for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFc.

(e) Affinity for vWF

FVIII interactions with von Willebrand factor (vWF) were measured byreal-time biomolecular interaction analysis (BIAcore), based on surfacePlasmon resonance (SPR) technology, to determine the kinetics of bindingof rFVIIIFc and rBDD FVIII towards vWF (TABLE 6). Kinetic rateparameters of K_(a) (on-rate) and K_(d) (off-rate), and the affinityK_(D) (K_(d)/K_(a)), were determined for each FVIII interaction underidentical conditions. Both rFVIIIFc and rBDD FVIII were found to have alow nM binding affinity (K_(D)) for vWF, of 1.64±0.37 and 0.846±0.181nM, respectively. The proteins had similar off-rates, with a two folddifference in on-rate resulting in a two fold difference in theaffinity.

TABLE 6 Biocore Binding Analysis of FVIII Proteins to vWF Kinetic rateparameters Off-rate/On-rate Analyte Ligand N On-rate (M−1s−1) Off-rate(s−1) KD(M) rFVIIIFc DS hvWf 5 7.92 ± 1.51 × 10⁵ 1.25 ± 1.12 × 10⁻³ 1.64± 0.37 × 10⁻⁹ NP rFVIIIFc hvWf 5 8.66 ± 1.10 × 10⁵ 1.09 ± 0.09 × 10⁻³1.28 ± 0.22 × 10⁻⁹ rBDD FVIII hvWf 5 13.7 ± 1.50 × 10⁵ 1.14 ± 0.12 ×10⁻³ 0.846 ± 0.181 × 10⁻⁹

As shown in TABLE 6, the affinity of rFVIIIFc DS or single chainrFVIIIFc with vWF was found to be in the low nM range, approximately twofold greater than that of BDD FVIII alone. At physiologicalconcentrations, this would result in a slight decrease in the percentageof rFVIIIFc (processed or single chain) complexed with vWF as comparedto free FVIII, however in vivo studies have indicated that the half-lifeof rFVIIIFc is significantly prolonged over full length or BDD FVIIIdespite this slightly lower affinity, and therefore this does not appearto compromise the half-life of the molecule. The free rFVIIIFc may bemore efficiently recycled through the FcRn pathway and thereforecontribute to a greater prolongation of half-life.

Example 3 rFVIIIFc Phase I/IIa Clinical Trial

A Phase I/IIa, open-label, crossover, dose-escalation, multi-center, andfirst-in-human study was designed to evaluate the safety, tolerability,and pharmacokinetics of a single dose of rFVIIIFc in subjects withsevere (defined as <1 IU/dL [1%] endogenous FVIII [FVIII]) hemophilia A.A total of approximately 12 previously treated patients were enrolledand dosed with rFVIIIFc at 25 or 65 IU/kg. After the screening(scheduled within 28 days prior to the first dose of the ADVATE®[rFVIII], the reference comparator agent) and a minimum of 4-days (96hours) elapsing with no FVIII treatment prior to the first injection,approximately 6 subjects received a single 25 IU/kg dose of ADVATE®followed by a 3-day (72 hours) pharmacokinetic (PK) profile thencrossover and receive a 25 IU/kg single, open-label dose of rFVIIIFc fora 7-day (168 hours) PK profiling. The first 3 subjects were dosedsequentially. For the first three (3) subjects dosed with 25 IU/kg ofrFVIIIFc, each subject underwent an inhibitor assessment at 14-days (336hours) post-injection of rFVIIIFc. Dosing of the next subject (for thefirst three subjects only) occurred once the inhibitor testing iscompleted. After the 3rd subject completed the 14 day inhibitorassessment, the remaining three subjects at 25 IU/kg and the sixsubjects at 65 IU/kg began enrollment sequentially at least 1 day apartwithin each dose group.

One week after the last subject received the 25 IU/kg dose of therFVIIIFc, approximately 6 unique subjects were recruited for the 65IU/kg cohort. Each subject in the 65 IU/kg cohort received a single 65IU/kg dose of ADVATE® followed by a 4-day (96 hours) PK profiling thencrossover and receive a 65 IU/kg single, open-label dose of rFVIIIFc fora 10-day (240 hours) profiling. If a bleeding episode occurred beforethe first injection of rFVIIIFc in any cohort, subject's pre-study FVIIIproduct was used for treatment and an interval of at least 4 days had topass before receiving the first injection of rFVIIIFc for the PKprofile.

All subjects were followed for a 14-day (336 hours) and 28 day safetyevaluation period after administration of rFVIIIFc 25 IU/kg or 65 IU/kgfor safety. All subjects underwent pharmacokinetic sampling pre- andpost-dosing along with blood samples for analysis of FVIII activity atdesignated time points.

The pharmacokinetic data for the Phase I/IIa clinical trial demonstratedthe following results for FVIIIFc. FVIIIFc had about a 50% increase insystemic exposure (AUC_(INF)), about 50% reduction in clearance (Cl),and about 50-70% increase in elimination half-life and MRT compared toADVATE® (full length rFVIII). In addition, FVIIIFc showed increasedC168, TBLP1, TBLP3, and TBLP5 values compared to ADVATE®.

The measured PK parameters were:

-   -   AUC_(INF) Area under the concentration-time curve from zero to        infinity    -   Beta HL Elimination phase half-life; also referred to as        t_(1/2β)    -   C168 Estimated FVIIIFc activity above baseline at approximately        168 h after dose    -   Cl Clearance    -   MRT Mean residence time    -   TBLP1 Model-predicted time after dose when FVIIIFc activity has        declined to approximately 1 IU/dL above baseline    -   TBLP3 Model-predicted time after dose when FVIIIFc activity has        declined to approximately 3 IU/dL above baseline    -   TBLP5 Model-predicted time after dose when FVIIIFc activity has        declined to approximately 5 IU/dL above baseline

Example 4 Pharmacokinetics (PK) of rFVIIIFc

A recombinant B-domain-deleted FVIII-Fc (rFVIIIFc) fusion protein hasbeen created as an approach to extend the half-life of FVIII. Thepharmacokinetics (PK) of rFVIIIFc were compared to rFVIII in hemophiliaA mice. The terminal half-life was twice as long for rFVIIIFc comparedto rFVIII. In order to confirm that the underlying mechanism for theextension of half-life was due to the protection of rFVIIIFc by FcRn,the PK were evaluated in FcRn knockout and human FcRn transgenic mice.

A single intravenous dose (125 IU/kg) was administered and the plasmaconcentration measured using a chromogenic activity assay. The C_(max)was similar between rFVIIIFc and rFVIII (XYNTHA®) in both mouse strains.However, while the half-life for rFVIIIFc was comparable to that ofrFVIII in the FcRn knockout mice, the half-life for rFVIIIFc wasextended to approximately twice longer than that for rFVIII in the hFcRntransgenic mice. These results confirmed that FcRn mediated or wasresponsible for the prolonged half-life of rFVIIIFc compared to rFVIII.Since hemostasis in whole blood measured by rotation thromboelastometry(ROTEM®) has been shown to correlate with the efficacy of coagulationfactors in bleeding models of hemophilia mice as well as in clinicalapplications, we sought to evaluate the ex vivo efficacy of rFVIIIFc inthe hemophilia A mice using ROTEM®.

Hemophilia A mice were administered a single intravenous dose of 50IU/kg rFVIIIFc, XYNTHA® (FVIII) or ADVATE® (FVIII). At 5 minutes postdose, clot formation was similar with respect to clotting time (CT),clot formation time (CFT) and α-angle. However, rFVIIIFc showedsignificantly improved CT at 72 and 96 hr post dose, and CFT and α-anglewere also improved at 96 hours compared to both XYNTHA® (FVIII) andADVATE® (FVIII), consistent with prolonged PK of rFVIIIFc. These resultsindicated that rFVIIIFC has a defined mechanism of action resulting inan increased half-life, and the potential to provide prolongedprotection from bleeding.

Example 5 rFVIIIFc Phase I/IIa Clinical Trial Results

This Example presents final analysis results for FVIII activity from 16patients treated with 25 and 65 IU/kg FVIII products. See Example 3.rFVIIIFc is a recombinant fusion protein comprised of a single moleculeof recombinant B-domain deleted human FVIII (BDD-rFVIII) fused to thedimeric Fc domain of the human IgG1, with no intervening linkersequence. This protein construct is also referred to herein as rFVIIIFcheterodimeric hybrid protein, FVIIIFc monomeric Fc fusion protein,FVIIIFc monomer hybrid, monomeric FVIIIFc hybrid, and FVIIIFcmonomer-dimer. See Example 1, FIG. 1, and TABLE 2A.

Preclinical studies with rFVIIIFc showed an approximately 2-foldprolongation of the half-life of rFVIII activity compared tocommercially available rFVIII products. The rationale for this study wasto evaluate the safety and tolerability of a single dose of rFVIIIFc infrozen liquid formulation and provide data on the PK in severehemophilia A subjects. For this study, 16 evaluable subjects wereavailable for PK evaluation. Single administration of two doses of bothrFVIIIFc and ADVATE® at a nominal dose of 25 (n=6) and 65 IU/kg of bodyweight (n=10) were infused intravenously over approximately 10 minutes.Blood samples for plasma PK assessments were obtained before infusion,as well as up to 10 days after dosing. The PK of FVIII activity for bothADVATE® and rFVIIIFc were characterized in this study using amodel-dependent method.

Objectives

The primary objective of this study was to assess the safety andtolerability of single administration of two doses of rFVIIIFc (25 and65 IU/kg) in previously treated patients (PTPs) aged 12 and above withsevere hemophilia A. The secondary objective was to determine thepharmacokinetics (PK) parameters determined by pharmacodynamic (PD)activity of FVIII over time after a single administration of 25 or 65IU/kg of rFVIIIFc compared to ADVATE® in one-stage clotting andchromogenic assays.

Study Design (see Example 3)

Blood samples were collected for FVIII activity PK evaluations at thescreening visit (within 28 days prior to dosing ADVATE®); on Day 0(injection of ADVATE®) pre-injection and at 10 and 30 minutes and 1, 3,6, and 9 hours post-injection; on Day 1 at 24 hours post-injection ofADVATE®; on Day 2 at 48 hours post-injection of ADVATE®; on Day 3 at 72hours post-injection of ADVATE®; and on Day 4 at 96 hours post-injectionof high dose of ADVATE® (Cohort B only).

Blood samples were collected for FVIII activity PK evaluations on theday of rFVIIIFc injection just prior to the administration of rFVIIIFc,at 10 and 30 minutes and 1, 3, 6, and 9 hours post-injection ofrFVIIIFc; on Day 1 at 24 hours post-injection of rFVIIIFc; on Days 2through 5 at 48, 72, 96, and 120 hours post-injection of rFVIIIFc; onDay 7 at 168 hours post-injection of rFVIIIFc; on Days 8, 9, and 10 at192, 216, and 240 hours post-injection of high dose of rFVIIIFc (CohortB only). FVIII activity was also measured at the final study visit (28days post-injection of rFVIIIFc) at 672 hours post-injection ofrFVIIIFc.

Pharmacokinetic Modeling

Abbreviations

-   -   TBLP1 Model-predicted time after dose when FVIII activity has        declined to approximately 1 IU/dL above baseline.    -   TBLP3 Model-predicted time after dose when FVIII activity has        declined to approximately 3 IU/dL above baseline        Calculations    -   KV_M=C_(max) _(_)M/Actual Dose (IU/kg)    -   KV_OB=C_(max) _(_)OB/Actual Dose (IU/kg)    -   IVR_M=100×C_(max) _(_)M×Plasma Volume (dL)/Total Dose in IU;        where plasma volume in mL=(23.7×Ht in cm)+(9.0×Wt in kg)−1709.    -   IVR_OB=100×C_(max) _(_)OB×Plasma Volume (dL)/Total Dose in IU;        where plasma volume in mL=(23.7×Ht in cm)+(9.0×Wt in kg)−1709.        Results

(a) Single-Dose Pharmacokinetics (One-Stage Assay)

Observed FVIII activity increased sharply after the short IV infusion ofeither ADVATE® or rFVIIIFc, with mean (±SD) model-predicted C_(max)values of 56.6±4.74 and 121±28.2 IU/dL for ADVATE® and 55.6±8.18 and108±16.9 IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. All ADVATE®- and rFVIIIFc-treated patients haddose-related increases in FVIII activity. The observed increase in bothC_(max) and AUC_(INF) was slightly less than proportional to dose overthe dose range evaluated.

After the end of the infusion, the decline of the observed FVIIIactivity exhibited monoexponential decay characteristics until thebaseline level was reached. The rate of decline in FVIII activity wasslower for rFVIIIFc than for ADVATE® with mean (±SD) model-predictedelimination half-life values of 11.9±2.98 and 10.4±3.03 hr for ADVATE®and 18.0±3.88 and 18.4±6.99 hr for rFVIIIFc for the 25 and 65 IU/kg dosegroups, respectively. Elimination half-life values appeared to bedose-independent over the dose range evaluated for both FVIII products.

Total systemic FVIII exposure (assessed by AUC_(INF)) was ˜48% and 61%greater following rFVIIIFc administration than ADVATE® at 25 and 65IU/kg dose levels, respectively. Mean (±SD) model-predicted AUC_(INF)values were 974±259 and 1810±606 hr*IU/dL for ADVATE® and 1440±316 and2910±1320 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively.

Similar to elimination half-life, the MRT was prolonged for rFVIIIFcrelative to ADVATE®. Mean (±SD) model-predicted MRT values were17.1±4.29 and 14.9±4.38 hr for ADVATE® and 25.9±5.60 and 26.5±10.1 hrfor rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRTvalues appeared to be dose-independent over the dose range evaluated forboth FVIII products.

In addition, primary PK parameter values for CL and V were determined.CL values for rFVIIIFc only accounted for ˜66% of those observed forADVATE® at equivalent doses. Mean (±SD) model-predicted CL values were2.70±0.729 and 4.08±1.69 mL/hr/kg for ADVATE® and 1.80±0.409 and2.69±1.25 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. V values were comparable between ADVATE® and rFVIIIFc withmean (±SD) model-predicted V values of 43.9±4.27 and 56.1±13.4 mL/kg forADVATE® and 45.3±7.23 and 61.6±10.6 mL/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively. Slight increases in mean CL and Vvalues were noted with increasing dose of ADVATE® and rFVIIIFc; however,the increase in standard deviations at the 65 IU/kg dose coupled withlimited dose levels confounded an assessment of the dose-dependency ofthese parameters. For example, the CV % geometric mean CL value for therFVIIIFc treatment group increased from 23.0% (25 IU/kg) to 48.6% (65IU/kg).

In addition to the primary PK parameters, secondary PK parameters (e.g.K-values, IVR, etc.) were determined to evaluate FVIII duration ofeffect. Evidence of PK difference was also observed with rFVIIIFcdemonstrating increased TBLP1 and TBLP3 values compared to ADVATE® atequivalent doses. IVR and K-values for ADVATE® and rFVIIIFc appeared tobe comparable. A slight increase in TBLP1 and TBLP3 values were observedwith increasing dose of ADVATE® and rFVIIIFc. In contrast, slightdecreases in mean IVR and K-values were noted with increasing dose ofADVATE® and rFVIIIFc. As previously indicated, an assessment of the dosedependency of these parameters is confounded by limited dose levels.

Mean (±SD) observed TBLP1 were 2.88±0.733 and 2.93±0.848 IU/dL per IU/kgfor ADVATE® and 4.28±0.873 and 5.16±2.02 IU/dL per IU/kg for rFVIIIFcfor the 25 and 65 IU/kg dose groups, respectively. Mean (±SD) observedTBLP3 were 2.06±0.527 and 2.26±0.666 IU/dL per IU/kg for ADVATE® and3.09±0.623 and 3.93±1.59 IU/dL per IU/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively.

Mean IVR and K-values calculated using observed C_(max) values(subtracted with baseline and residual drug within the model) weregenerally greater than values determined using model-predicted C_(max)values; consistent with slight underestimation of the observed peakactivity using the one-compartment model. Mean (±SD) observed K-valueswere 2.57±0.198 and 2.13±0.598 IU/dL per IU/kg for ADVATE® and2.46±0.330 and 1.85±0.332 IU/dL per IU/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively. Mean (±SD) observed IVR values were94.1±15.6 and 85.8±16.5% for ADVATE® and 89.5±11.9 and 74.8±6.72% forrFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

(b) Single-Dose Pharmacokinetics (Chromogenic Assay)

Observed FVIII activity increased sharply after the short IV infusion ofeither ADVATE® or rFVIIIFc, with mean (±SD) model-predicted C_(max)values of 70.2±9.60 and 157±38.6 IU/dL for ADVATE® and 70.3±10.0 and158±34.7 IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively.

All ADVATE®- and rFVIIIFc-treated patients had dose-related increases inFVIII activity. The observed increase in both C_(max) and AUC_(INF) wasslightly less than proportional to dose over the dose range evaluated.

After the end of the infusion, the decline of the observed FVIIIactivity exhibited monoexponential decay characteristics until thebaseline level was reached. The rate of decline in FVIII activity wasslower for rFVIIIFc than for ADVATE® with mean (±SD) model-predictedelimination half-life values of 10.7±1.98 and 10.3±3.27 hr for ADVATE®and 16.2±2.92 and 19.0±7.94 hr for rFVIIIFc for the 25 and 65 IU/kg dosegroups, respectively. Elimination half-life values appeared to bedose-independent over the dose range evaluated for both FVIII products.

Total systemic FVIII exposure (assessed by AUC_(INF)) was ˜53% and 84%greater following rFVIIIFc administration than ADVATE® at 25 and 65IU/kg dose levels, respectively. Mean (±SD) model-predicted AUC_(INF)values were 1080±236 and 2320±784 hr*IU/dL for ADVATE® and 1650±408 and4280±1860 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively.

Similar to elimination half-life, the MRT was prolonged for rFVIIIFcrelative to ADVATE®. Mean (±SD) model-predicted MRT values were15.3±2.86 and 14.8±4.72 hr for ADVATE® and 23.4±4.22 and 27.3±11.4 hrfor rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRTvalues appeared to be dose-independent over the dose range evaluated forboth FVIII products.

In addition, primary PK parameter values for CL and V were determined.CL values for rFVIIIFc only accounted for ˜58-66% of those observed forADVATE® at equivalent doses. Mean (±SD) model-predicted CL values were2.39±0.527 and 3.21±1.40 mL/hr/kg for ADVATE® and 1.57±0.349 and1.86±0.970 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. V values were comparable between ADVATE® and rFVIIIFc withmean (±SD) model-predicted V values of 35.8±5.52 and 43.6±11.2 mL/kg forADVATE® and 35.9±6.65 and 42.7±8.91 mL/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively. Increases in mean CL and V values werenoted with increasing dose of ADVATE® and rFVIIIFc; however, theincrease in standard deviations at 65 IU/kg coupled with limited doselevels confounded an assessment of the dose-dependency of theseparameters.

In addition to the primary PK parameters, secondary PK parameters (e.g.K-values, IVR, etc.) were determined to evaluate FVIII duration ofeffect. Evidence of PK difference was also observed with rFVIIIFcdemonstrating increased TBLP1 and TBLP3 values compared to ADVATE® atequivalent doses. IVR and K-values for ADVATE® and rFVIIIFc appeared tobe comparable.

A slight increase in TBLP1 and TBLP3 values were observed withincreasing dose of ADVATE® and rFVIIIFc. In contrast, slight decreasesin mean IVR and K-values were noted with increasing dose of ADVATE® andrFVIIIFc. As previously indicated, an assessment of the dose dependencyof these parameters is confounded by limited dose levels.

Mean (±SD) observed TBLP1 were 2.70±0.511 and 3.09±0.978 IU/dL per IU/kgfor ADVATE® and 4.06±0.798 and 5.66±2.38 IU/dL per IU/kg for rFVIIIFcfor the 25 and 65 IU/kg dose groups, respectively. Mean (±SD) observedTBLP3 were 1.98±0.377 and 2.39±0.718 IU/dL per IU/kg for ADVATE® and3.04±0.598 and 4.44±1.84 IU/dL per IU/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively.

Mean IVR and K-values calculated using observed C_(max) values(subtracted with baseline and residual drug within the model) weregenerally greater than values determined using model-predicted C_(max)values; consistent with slight underestimation of the observed peakactivity using the one-compartment model. Mean (±SD) observed K-valueswere 3.08±0.429 and 2.85±0.721 IU/dL per IU/kg for ADVATE® and3.12±0.451 and 2.92±0.985 IU/dL per IU/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively. Mean (±SD) observed IVR values were112±14.5 and 116±26.9% for ADVATE® and 113±16.3 and 117±33.6% forrFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

CONCLUSIONS

All ADVATE®- and rFVIIIFc-treated patients had comparable dose-relatedincreases in C_(max) and AUC_(INF) over the dose range evaluated. Peakplasma levels of ADVATE® and rFVIIIFc activity were generally observedwithin the first hour after the end of the infusion and remaineddetectable for several days after dosing. After the end of infusion, thedecline in baseline corrected FVIII activity exhibited monoexponentialdecay until the baseline was reached for both products. Parameter valuesfor elimination half-life and MRT appeared to be dose-independent overthe dose range evaluated for both FVIII products. Slight increases inmean CL and V values were noted with increasing dose of ADVATE® andrFVIIIFc; however, increased intersubject variability at the 65 IU/kgcoupled with limited dose levels confounded an assessment of thedose-dependency of these parameters.

Comparison of rFVIIIFc and ADVATE® activity PK revealed an approximate48-61% (One-Stage Assay) or 53-84% (Chromogenic Assay) increase insystemic exposure, approximate 30-40% reduction in clearance, and anapproximate 50-80% increase in both elimination half-life and MRT forrFVIIIFc relative to ADVATE® at comparable doses. Evidence of PKdifference was also observed with rFVIIIFc demonstrating increased TBLP1and TBLP3 values compared to ADVATE® at equivalent doses. IVR andK-values for ADVATE® and rFVIIIFc appeared to be comparable.

The PK parameters obtained from the Chromogenic Assay results generallyagreed with those from the One-Stage Assay, except that the ChromogenicAssay yielded a higher estimation of exposure parameters (e.g., C_(max),AUC_(INF), etc.).

The observed improvements in PK, indicate that rFVIIIFc can provideprolonged protection from bleeding, allowing less frequent injectionsfor individuals with Hemophilia A.

Example 6 rFVIIIFc A-LONG Phase 3 Clinical Study

On the basis of the interim PK analysis from the first in-human study ofrFVIIIFc (see Example 3), the A-LONG study was designed. A-LONG was anopen label, global, multi-center, Phase 3 evaluation of the safety,pharmacokinetics, and efficacy of recombinant FVIII Fc fusion (FVIII:Fc)in the prevention and treatment of bleeding in previously treatedsubjects with severe hemophilia A (defined as <1 IU/dL [<1%] endogenousFVIII).

The primary objectives of the A-LONG study were (i) to evaluate safetyand tolerability of rFVIIIFc administered as prophylaxis, weekly,on-demand, and surgical treatment regimens, and (ii) to evaluate theefficacy of rFVIIIFc administered as tailored prophylaxis, on-demand,and surgical treatment regimens. The secondary objective of the A-LONGstudy were (i) to characterize the PK profile of rFVIIIFc and comparethe PK of rFVIIIFc with the currently marketed product, ADVATE®, (ii) toevaluate individual responses with rFVIIIFc, (iii) to characterize therange of dose and schedules required to adequately prevent bleeding in aprophylaxis regimen, maintain homeostasis in a surgical setting, or totreat bleeding episodes in an on-demand, weekly treatment, orprophylaxis setting, and (iv) to evaluate rFVIIIFc consumption (e.g.,total annualized rFVIIIFc consumption per subject).

165 subjects were enrolled into one of three regimens: a tailoredprophylaxis regimen (Arm 1), a weekly dosing regimen (Arm 2), and anon-demand regimen (Arm 3). In addition, rFVIIIFc was evaluated in aperioperative management subgroup.

Key Inclusion Criteria:

(i) male, (ii) ≥12 years of age and at least 40 kg, (iii) diagnosis ofsevere hemophilia A defined as <1% (<1 IU/dL) endogenous FVIII activity,and (iv) history of ≥150 prior documented exposure days with anycurrently marketed FVIII product.

Arm 1: Tailored Prophylaxis Regimen

Arm 1 included an overall group and a PK subgroup. The initial regimenwas twice weekly at 25 IU/kg on the first day, followed by 50 IU/kg onthe fourth day of the week (Day 4). Subjects were administered rFVIIIFcon this weekly prophylaxis regimen until PK results for rFVIIIFc wereavailable. Based on these results, a tailored prophylaxis regimen wasestablished for each individual, in which the dose and interval wasdetermined to maintain a trough level of 1-3% FVIII activity. Eachsubject was then administered his individually tailored prophylaxisregimen throughout the study.

Subjects were monitored throughout the study and ongoing dose andinterval adjustments were made. Adjustments were only made when asubject experienced unacceptable bleeding episodes defined as ≥2spontaneous bleeding episodes over a rolling two-month period. In thiscase, adjustment targeted trough levels of 3-5%.

Arm 2: Weekly Dosing Regimen

Subjects underwent abbreviated rFVIIIFc PK profiling as follows: Washoutof at least 96 hours; a single dose of rFVIIIFc 65 IU/kg; Abbreviatedsampling beginning on rFVIIIFc Day 0, including pre-injection and 10(±2) minutes, 3 hours (±15 minutes), 72 (±2) hours [Day 3], and 96 (±2)hours [Day 4] from the start of injection. Following the abbreviated PKprofiling, subjects were then administer a fixed dose of 65 IU/kgrFVIIIFc every 7 days at least for 28 weeks and up to 52 weeks.

Arm 3: Episodic (On-Demand) Treatment

Subjects received rFVIIIFc episodic treatment as needed for bleeding.Subjects were enrolled and randomized and underwent abbreviated rFVIIIFcPK profiling as follows:

-   -   (i) Washout: At least 96 hours.    -   (ii) Dosing at rFVIIIFc Day 0: A single dose of rFVIIIFc 50        IU/kg administered under medical supervision.    -   (iii) Abbreviated sampling beginning at rFVIIIFc Day 0:        Preinjection and 30 (±3) minutes, 3 hours (±15 minutes), 72 (±2)        hours [Day 3], and 96 (±2) hours [Day 4] from the start of the        injection.

At the selection sites, sampling for TGA was done coincident with all PLprofiling time points. For the subset of subjects undergoing samplingfor ROTEM/TEG, collections were done at the following time points:Preinjection and 3 hours (±15 minutes), 72 (±2) hours [Day 3], and 96(±2) hours [Day 4] from the start of the injection.

Between scheduled visits, the subjects treated bleeding episodes atrFVIIIFc doses between 10 and 50 IU/kg, depending on the severity of thebleeding.

Perioperative Management Subgroup

rFVIIIFc was administered prior to a following surgery in the subset ofpatients requiring a major surgical procedure during the study. Majorsurgery is defined as any surgical procedure (elective or emergent) thatinvolves general anesthesia and/or respiratory assistance in which amajor body cavity is penetrated and exposed, or for which a substantialimpairment of physical or physiological functions is produced (e.g.,laparotomy, thoracotomy, craniotomy, joint replacement, and limbamputation).

For prophylaxis during surgery, subjects were treated with 20 to 50IU/kg rFVIIIFc every 12 to 24 hours. Prior to surgery, the physicianreviewed the subject's rFVIIIFc PK profile and assessed the dose regimenof FVIII replacement generally required for the type of planned surgeryand the clinical status of the subject. Recommendation for theappropriate dosing of rFVIIIFc in the surgical treatment period,including any rehabilitation time, took these factors intoconsideration.

Pharmacokinetic (PK) Assessment:

All subjects in all arms had an initial PK assessment after their firstdose of rFVIIIFc. A subset of subjects from Arm 1 were assigned to aprotocol-specified sequential PK subgroup to compare the PK of rFVIIIFcwith recombinant factor VIII (rFVIII, ADVATE® [anti-hemophilic factor(recombinant) plasma/albumin-free method]) as follows:

-   -   (i) Prior to treatment in Arm 1, PK was assessed after a single        dose of ADVATE® 50 IU/kg. PK was then assessed in these same        subjects after a single dose of rFVIIIFc 50 IU/kg.    -   (ii) PK of rFVIIIFc was repeated at 12 to 24 weeks.

Key Efficacy Outcome Measures (Included in Initial Readout):

(i) Annualized bleeding rate (ABR) in Arm 1 versus Arm 3 (individualizedprophylaxis arm compared with episodic treatment arm), (ii) number ofinjections required to resolve a bleeding episode, (iii) treatingphysicians' assessments of subjects' response to surgery with rFVIIIFcusing a 4-point scale.

PK Outcome Measures:

PK of rFVIIIFc and ADVATE®.

Key Safety Outcome Measures:

(i) Incidence of inhibitor development; and, (ii) incidence of adverseevents (AEs) occurring outside of the perioperative management period.

Results:

Subjects:

A total of 165 subjects were enrolled in the study. Arm 1(individualized prophylaxis), n=118; Arm 2 (weekly prophylaxis), n=24;Arm 3 (episodic treatment), n=23; Perioperative management subgroup,n=9, 9 surgeries (8 subjects from Arm 1, and 1 from Arm 2). 92.7% ofsubjects completed the study.

Efficacy:

Median ABR (individualized prophylaxis arm: 1.6; weekly prophylaxis arm:3.6; episodic treatment arm: 33.6). In the individualized prophylaxisarm, the median dosing interval was 3.5 days during the last 3 months onstudy. 30 percent of patients in the individualized prophylaxis armachieved a mean dosing interval of at least 5 days. 98% of bleedingepisodes were controlled by one or two injections of rFVIIIFc. Inperioperative management, treating physicians rated the hemostaticefficacy of rFVIIIFc as excellent or good in 100% of surgeries.

PK:

The geometric mean terminal half-life of rFVIIIFc was approximately 19.0hours, which is 1.53-fold longer than that of ADVATE® (approximately12.4 hours).

Safety:

No inhibitors were detected to rFVIIIFc, and no cases of anaphylaxiswere reported. rFVIIIFc was generally well tolerated. The most commonAEs, regardless of causality, (incidence ≥5%) occurring outside of theperioperative management period were nasopharyngitis, arthralgia,headache, and upper respiratory tract infection. 12 subjects (7.3%)experienced at least one serious AE (SAE) outside of the perioperativemanagement period. No SAEs were assessed to be related to drug by theinvestigator.

SUMMARY

Individualized and weekly prophylactic regimens resulted in lowsingle-digit median annualized bleeding rates. In the individualizedprophylaxis arm, the median dosing interval was 3.5 days. During thelast 3 months on study, 30 percent of patients in the individualizedprophylaxis arm achieved a mean dosing interval of at least 5 days. 98%of bleeding episodes were controlled by one or two injections ofrFVIIIFc. Hemostatic efficacy of rFVIIIFc during surgery was rated bytreating physicians as excellent or good in 100% of surgeries. Thehalf-life of rFVIIIFc was approximately 19.0 hours compared to 12.4hours for ADVATE®. No subject developed an inhibitor or experienced ananaphylactic reaction to rFVIIIFc. Recombinant FVIIIFc was generallywell tolerated.

Example 7 Clinical ROTEM® Assessment

In addition to the measurement of plasma FVIII activity by one-stageactivated partial thromboplastin time (aPTT) assay, whole bloodrotational thromboelastometry (ROTEM®) has also been explored to assessthe improvement in global hemostasis by rFVIIIFc and ADVATE® in 2subjects, specifically, 1 in the low dose cohort and 1 in the high dosecohort.

rFVIIIFc and ADVATE® appear to be comparably active in clot formationwhen spiked into subjects' blood prior to rFVIIIFc treatment. Theclotting time (CT) was linear with respect to the dose of rFVIIIFc andADVATE® in the range of approximately 1% of 100% of normal, and the doseresponse was comparable between rFVIIIFc and ADVATE® in the samesubject.

Following dosing with ADVATE® and subsequently rFVIIIFc, citrated wholeblood was sampled at various time points and the clot formationfollowing recalcification was monitored by ROTEM®. Despite the variablebaseline CT due to residue FVIII levels prior to ADVATE® or rFVIIIFcdosing, both products effectively corrected the CT to comparable levels30 minutes post-injection. In addition, the improvement in CT was bettersustained at and after 3 hours post-injection of 25 IU/kg of rFVIIIFcrelative to ADVATE® in the subject dosed at this low dose. However, thedifferential improvement of rFVIIIFc versus ADVATE® was much lessappreciable at the 65 IU/kg dose.

Example 8 In Vivo Efficacy of rFVIIIFc and Single Chain (SC) rFVIII inHemA Mice

Recombinant Factor VIIIFc (rFVIIIFc) is comprised of a B domain deleted(BDD) rFVIII protein genetically fused to the Fc domain of humanimmunoglobulin G1 (IgG1). Prior to secretion from HEK 293 cells, most ofthe rFVIIIFc is processed into a FVIII heavy chain (HC) and light chain(LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrandfactor (VWF) and released upon activation in a manner that isindistinguishable from native FVIII. Spontaneous dissociation of the HCand LC is thought to contribute to the loss of FVIII activity in plasmaand during storage of FVIII drug products. Here we describe a singlechain non-processed isoform of rFVIIIFc (SC rFVIIIFc), which can providesuperior manufacturability and enhanced stability compared to nativeFVIII.

SC rFVIIIFc was purified from rFVIIIFc, which contains a fraction of thenon-processed isoform. Compared to rFVIIIFc, SC rFVIIIFc showedequivalent chromogenic activity but approximately 60% reduced activityby the one stage (aPTT) assay, (TABLES 3A and 3B). Thrombin generationassay (TGA) was performed using calibrated automated thrombogram (fromThrombinoscope®). In a thrombin generation assay (TGA), SC rFVIIIFc alsoshowed a reduced thrombin potential (FIG. 13A), and peak thrombin (FIG.13B) compared to rFVIIIFc. However, as shown in TABLE 3B, full activityof SC rFVIIIFc by aPTT was observed in the absence of vWF, suggestingrelease from vWF can be delayed due to covalent linkage of the a3 acidicregion to the HC after Arg 1680 cleavage in SC rFVIIIFc, in contrast toa3 release and dissociation from fully processed FVIII. Delayeddissociation from vWF can explain the reduced activity observed in theaPTT assay and TGA, while full activity was observed in the two-stagechromogenic assay. A reduced rate of activation in the presence of vWFwas confirmed in a modified chromogenic substrate assay with limitingthrombin as FVIII activator.

In vivo function of SC rFVIIIFc was assessed in the HemA mouse tail veintransection (TVT) model. The mice were first anesthetized and theninjected with 4.6 μg/kg, 1.38 μg/kg, or 0.46 μg/kg of either processedrFVIIIFc (Drug Substance, which contain about 75%-85% processedrFVIIIFc) and purified single chain (SC) rFVIIIFc 48 hours prior to TVT.The tail was cut from the tip and immediately placed into a tube tocollect blood. Percentage of protection on survival was measured forrFVIIIFc processed (drug substance) and single chain (SC) FVIIIFc asshown in TABLE 7 and FIGS. 7A, 7B and 7C.

TABLE 7 In vivo Efficacy of rFVIIIFc DS and Single Chain (SC) rFVIIIFcDose (μg/kg) 4.6 1.38 0.46 % of Protection FVIIIFc DS 93 52 19 onSurvival Single chain 93 64 14 rFVIIIFc

In vivo Tail re-bleeding and survival were monitored hourly up to 12hours post TVT with final observation performed at 24-hour post TVT. SCrFVIIIFc and the rFVIIIFc demonstrated equivalent in vivo efficacy inthis model, with an ED50 of 1.17 μg/kg and 1.23 μg/kg respectively whenTVT was performed at 48 hours post infusion (FIG. 7A). Comparable 24hour post TVT survival curves (p≥0.65) (FIG. 7B) and re-bleed rates(FIG. 7C) in HemA mice were observed for the SC rFVIIIFc and rFVIIIFc ateach tested dose level, indicating that SC rFVIIIFc was equallyeffective as rFVIIIFc despite its lower apparent aPTT activity. Thedelayed in vitro activation of SC rFVIIIFc in the presence of vWFtherefore appeared to have no significant impact on its in vivoefficacy. These observations indicated that SC rFVIIIFc represents anovel and efficacious isoform of rFVIIIFc with potential clinicalapplications.

Example 9 Phase 1/2a Study Clinical Trial

rFVIIIFc is a recombinant fusion protein composed of a single moleculeof FVIII covalently linked to the Fc domain of human IgG₁ to extendcirculating rFVIII half-life. This first-in-human study inpreviously-treated male subjects with severe hemophilia A investigatedsafety and pharmacokinetics of rFVIIIFc. Sixteen subjects received asingle dose of ADVATE® at 25 or 65 IU/kg followed by an equal dose ofrFVIIIFc. Most adverse events were unrelated to study drug. None of thestudy subjects developed anti-FVIIIFc antibodies or inhibitors. Acrossdose levels, as compared with ADVATE®, rFVIIIFc showed 1.54 to 1.71-foldlonger elimination t_(1/2) and mean residence time, 1.49 to 1.56-foldlower clearance, and 1.48 to 1.56-fold higher total systemic exposure.ADVATE® and rFVIIIFc had comparable dose-dependent peak plasmaconcentrations and recoveries. Time to 1% FVIII activity above baselinewas approximately 1.53 to 1.68-fold longer than ADVATE® across doselevels. Thus, rFVIIIFc can offer a viable therapeutic approach toachieve prolonged hemostatic protection and less frequent dosing inpatients with hemophilia A.

rFVIIIFc is a recombinant fusion protein composed of a single moleculeof B-domain deleted rFVIII covalently linked to the human IgG₁ Fcdomain. Potential advantages of Fc-fusion proteins include bettertolerability and prolonged hemostatic protection, and the Fc domainrepresents a natural molecule with no known inherent toxicity. Dumont J.A. et al., BioDrugs 20(3):151-60 (2006), Dumont J. A. et al., “MonomericFc fusion technology: an approach to create long-lasting clottingfactors,” in: Kontermann R., ed., Therapeutic Proteins—Strategies toModulate Half-Life, Chapter 11, Wiley VCH publisher; prepublishedonline, DOI: 10.1002/9783527644827.ch10. Attachment to the IgG₁ Fcdomain permits binding to the neonatal Fc receptor (FcRn), which isexpressed in many cell types, including endothelial cells. FcRnexpression remains stable throughout life and is responsible forprotecting IgG₁ and Fc-fusion proteins from lysosomal degradation, thusprolonging the t_(1/2) of the protein. Dumont J. A. et al., BioDrugs20(3): 151-60 (2006), Roopenian D. C. et al., Nat Rev Immunol.7(9):715-25 (Epub 2007 Aug. 17). Numerous proteins within thecirculation are internalized into the cells lining the vasculature vianonspecific pinocytosis and are trafficked to endosomal and lysosomaldegradation pathways.

Fc proteins interact with FcRn, resident within endosomes. Endosomescontaining FcRn direct the Fc fusion proteins back to the plasmamembrane, releasing them into circulation in a pH-dependent manner,Lencer W. I. and Blumberg R. S., Trends Cell Biol. 15(1):5-9 (2005)thereby avoiding lysosomal degradation. This recycling approach has beenused successfully to extend the t_(1/2) of therapeutic biologics; anumber of Fc fusion-based drugs have been approved for clinical use(e.g., etanercept, romiplostim) and others are in development. Huang C.,Curr Opin Biotechnol. 20(6):692-9. (Epub 2009 Nov. 4), Schmidt S. R.,Curr Opin Drug Discov Devel. 12(2):284-295 (2009).

Preclinical data for rFVIIIFc indicate that FVIII can be rescued fromdegradation by a natural protective pathway mediated by FcRn, thusextending t_(1/2). In Hemophilia A mice and dogs, terminal plasmat_(1/2) for rFVIIIFc was approximately 2 times longer than with rFVIII.Dumont J. et al., Blood. 116(21) Abstract 545 (2009), Liu T. et al., JThromb Haemost. 9(52):561 (2011). Based on these data, we conducted afirst-in-human clinical study to investigate the safety and PK of along-lasting rFVIIIFc fusion protein in subjects with hemophilia A.

Study Design

This open-label, dose-escalation, multicenter Phase 1/2a study inpreviously treated patients with severe hemophilia A investigated thesafety of rFVIIIFc and its pharmacokinetics (PK) compared with ADVATE®(antihemophilic factor [recombinant], plasma/albumin-free method,octocog alfa, Baxter Healthcare). This study was performed in accordancewith the US CFR and ICH Guidelines on Good Clinical Practices. Prior toany testing, approval from participating Institutional Review Boards andwritten informed consents from all subjects were obtained. The studydesign was sequential; a single dose of ADVATE® was administered at 25or 65 IU/kg followed by an equal dose of rFVIIIFc (FIG. 8). Both drugswere injected intravenously over approximately 10 minutes. The two doselevels were expected to bracket the typical therapeutic dose ranges.Subjects were followed for 28 days after receiving rFVIIIFc for safetyanalyses, including testing for anti-FVIII antibodies and inhibitors at14 and 28 days post-injection. Plasma FVIII activity was measured insubjects before injection, 10 and 30 minutes, 1, 3, 6, 9, 24, 48, 72,96, 120, and 168 hours (7 days) after rFVIIIFc injection, withadditional samples at 192, 216, and 240 hours (10 days) for subjectsdosed at 65 IU/kg of rFVIIIFc. Plasma FVIII activity was measured at thesame time points after ADVATE® treatment, through 72 hours for the 25IU/kg group and 96 hours for the 65 IU/kg group.

(a) Subjects

Male subjects were at least 12 years of age with severe hemophilia A(defined as FVIII activity level <1%) and had at least 100 documentedprior exposure days to FVIII concentrates (pdFVIII or rFVIII). Subjectswith known hypersensitivity to mouse or hamster protein, history ofinhibitor or detectable inhibitor titer at screening, or who were takingany medications that could affect hemostasis or systemicimmunosuppressive drugs, or who experienced an active bacterial or viralinfection (other than hepatitis or HIV) within 30 days of screening wereexcluded. Subject's genotype was recorded at study entry, when known.

(b) Treatment Product

The human rFVIIIFc and Fc transgenes were stably transfected into HEK293cells and the cell line was extensively tested for stability, sterility,and viral contamination to ensure safety. The purified drug product iscomposed of a monomeric B-domain-deleted FVIII covalently linked throughits carboxy-terminus to the N-terminus of an Fc monomer, which forms adisulfide bond with a second Fc monomer during synthesis and secretionfrom the cells. rFVIIIFc was purified by chromatography andnanofiltration, and was fully active in one-stage and chromogenicclotting assays relative to commercially available rFVIII preparations.It was supplied as a frozen liquid containing 1000 IU per 2 mL ofsolution and formulated with L-histidine (pH 7), sodium chloride,calcium chloride, sucrose, mannitol, and Polysorbate 20. For injection,the product was diluted with saline solution (0.9% NaCl).

(c) Outcome Measures

The primary objective of the study was safety, evaluated throughphysical examination, reporting of treatment-emergent adverse events(AEs), development of antibodies, and laboratory monitoring over time.The secondary objectives included parameters derived from PK analyses.Laboratory assessments included prothrombin time, activated partialthromboplastin time (aPTT), international normalized ratio, levels ofD-dimer, von Willebrand factor (vWF) antigen, standard hematology andblood chemistry tests, and urinalysis.

FVIII activity was measured by the one-stage clotting (aPTT) assay on aSiemens BCS-XP analyzer using commercial reagents (Dade Actin FSL) withcalibration against a normal reference plasma (Precision BiologicsCRYOcheck™) traceable to the World Health Organization (WHO) 5^(th)International Standard (IS) for human plasma. In addition to the aPTTassay, FVIII activity was measured by a chromogenic substrate assayRosen S., Scand J Haematol Suppl. 33(Suppl 40):139-45 (1984) using acommercially available kit (Aniara BIOPHEN FVIII:C) that complies withEuropean Pharmacopoeia recommendations. The chromogenic assay wascalibrated against normal human reference plasma (InstrumentationLaboratories ORKE45), which also had a potency assigned against the WHO5th IS human plasma standard.

The lower limit of quantification (LLOQ) for the one-stage andchromogenic assays was 0.5 IU/dL and 0.4 IU/dL, respectively. FVIIIinhibitors were measured by the Nijmegen-modified Bethesda assay andless than 0.6 BU/mL was considered negative. Anti-rFVIIIFc antibodieswere assessed using a specific bridging electrochemiluminescentimmunoassay which uses biotin and sulfo-tagged rFVIIIFc. Assaysensitivity was determined to be 89 ng/mL using an anti-human FVIIImonoclonal antibody as a surrogate control. Exploratory whole bloodrotation thromboelastometry (ROTEM®) was performed in two subjects, onefrom each dose level, at various time points to assess the improvementin global hemostasis following injection with ADVATE® and rFVIIIFc.

(d) Pharmacokinetic Analyses

A user-defined one-compartment disposition model, which automaticallyestimates the endogenous FVIII level and subsequent residual decay, wasutilized in WINNONLIN® for analysis of the individual subject plasmaFVIII activity-versus-time data following a single administration ofADVATE® or rFVIIIFc. Actual sampling times, doses, and duration ofinjection were used for calculations of parameters including maximumactivity (C_(max)), t_(1/2), clearance (CL), volume of distribution atsteady-state (V_(ss)), area under the curve (time zero extrapolated toinfinity [AUC_(INF)]), mean residence time (MRT), and incrementalrecovery.

Monte Carlo Simulation of rFVIIIFc Activity-Versus-Time Profile:

To construct FVIII activity-time profiles following dosing regimens of25 IU/kg or 65 IU/kg, a Monte Carlo simulation was conducted using thepopulation PK model of ADVATE® and rFVIIIFc. The mean estimates of modelparameters (CL, volume of distribution) in the tested population, theinter-individual variance, and the residual variability were estimatedbased on the one-stage (aPTT) clotting assay activity data of ADVATE®and rFVIIIFc from 16 subjects in this Phase1/2a study. Five hundredsubjects were simulated with 15 sampling points for each subject foreach dosing regimen. The percentage of the population with FVIIIactivity above or equal to 1% and 3% at different times followingdifferent dosing regimens of ADVATE® or rFVIIIFc was estimated.

Statistical Analyses:

Selected PK parameters for rFVIIIFc and ADVATE® were compared using ananalysis of variance model. PK parameters were log-transformed for theseanalyses and estimated means, mean differences, and confidence intervalson the log-scale were transformed to obtain estimates for geometricmeans, geometric mean ratios (GMR), and confidence intervals,respectively, on the original scale. The GMR is the geometric mean ofthe intra-subject ratio of the rFVIIIFc PK parameter value to theADVATE® PK parameter value.

Results

Subject Disposition:

Nineteen subjects were enrolled in the study; 16 underwent PK evaluationfor both ADVATE® and rFVIIIFc. One subject self-administered hisprevious product prior to completing the wash-out period following thedose with ADVATE® and was thus excluded from the PK analysis, but wasincluded in the safety analysis. Three subjects were discontinued fromthe study before receiving either study drug: one voluntarily withdrew;a second was withdrawn by the Investigator for non-compliance; and onewas withdrawn at the Sponsor's request due to completion of studyenrollment. Of the subjects dosed, six subjects received 25 IU/kg and 10subjects received 65 IU/kg of both ADVATE® and rFVIIIFc. Mean age was40.3 years (23 to 61 years). Genotypic identification was collected forseven subjects; inversion of intron 22 was reported in six subjects; anda frame-shift defect was reported in one subject. The genotype wasunknown for nine subjects. Thirteen subjects had hepatitis C antibodies,four of whom were also positive for HIV.

Safety:

Forty-four treatment-emergent AEs were reported by 11 (69%) subjectsduring the treatment and follow-up periods. This included the day ofdosing with ADVATE® or rFVIIIFc through a 28-day post-dosing observationperiod. The majority of events were considered mild and none led towithdrawal from the study. One event, dysgeusia, occurred transiently inone subject while receiving a 65 IU/kg dose of rFVIIIFc and wasconsidered related to rFVIIIFc. One subject experienced an anxietyattack after receiving 65 IU/kg of rFVIIIFc which resulted in 21 AEs, 19of which were graded as mild, and two of which (headache andphotophobia) were rated as moderate. Neither was deemed related torFVIIIFc by the Investigator. No serious bleeding episodes werereported. No evidence of allergic reactions to injection was detected.All plasma samples tested negative for FVIII inhibitors andanti-rFVIIIFc antibodies. No signs of injection site reactions wereobserved. No clinically meaningful changes in abnormal laboratory valueswere reported.

Pharmacokinetics:

Correlation Between aPTT and Chromogenic Activity for rFVIIIFc inPlasma:

ADVATE® and rFVIIIFc activities were determined in the same assays usingcommercially available reagents and calibration against normal humanplasma standards. There was a strong correlation between the resultsobtained by the one-stage clotting assay and the chromogenic assay insamples that had an activity above the LLOQ. Correlation coefficients(Pearson R²) of 0.94 and 0.95 were observed between the two assayresults for 151 samples following ADVATE® dosing and 185 samplesfollowing rFVIIIFc dosing, respectively. Compared to the aPTT results,the chromogenic FVIII activities were, on average, 21% higher forADVATE® and 32% higher for rFVIIIFc, not statistically significant (FIG.9). This observation led to a slightly higher estimation of exposureparameters by the chromogenic assessment for both drugs. The apparenthigher FVIII recoveries by the chromogenic assay are typical forrecombinant FVIII products tested in clinical assays, and are inagreement with most other marketed FVIII products. Lee C. A. et al.,Thromb Haemost. 82(6):1644-7 (December 1999), Mikaelsson M. andOswaldsson U., Semin Thromb Hemost. 28(3):257-64 (June 2002), StroobantsA. K. et al., J Thromb Haemost. 9 (Suppl 2) (2011).

Improved Pharmacokinetics for rFVIIIFc:

The primary PK estimates were derived from one-stage (aPTT) clottingassay activity data. In subjects who received 25 or 65 IU/kg of ADVATE®followed by an equal dose of rFVIIIFc, the plasma FVIII activity rosesharply and reached C_(max) within the first hour following dosing. Thesubsequent decline of the observed FVIII activity exhibitedmonoexponential decay characteristics until the baseline FVIII activitywas reached (FIGS. 10A and 10B). The C_(max) increased proportionally tothe dose, but was comparable between equal doses of ADVATE® and rFVIIIFc(TABLE 8). The total exposure (AUC_(INF)) also increased proportionallyto the dose. However, the AUC_(INF) of rFVIIIFc was 1.48 and 1.56-foldgreater than that of ADVATE® at 25 IU/kg (p=0.002) and 65 IU/kg(p<0.001), respectively (TABLE 8).

TABLE 8 PK Parameters by One-Stage (aPTT) Assay for rFVIIIFc andADVATE ® Per Dose Group Dose: 25 IU/kg (N = 6) Dose: 65 IU/kg (N = 9)ADVATE ® rFVIIIFc Geom. ADVATE ® Geom. Geom. Geom. Mean Ratio Geom.rFVIIIFc Mean Ratio Mean Mean [95% CI] Mean Geom. Mean [95% CI]Parameter [95% CI] [95% CI] (p-value) [95% CI] [95% CI] (p-value)C_(max) _(—) OBS 63.6 60.5 0.952 133 119 0.895 (IU/dL) [59.1, 68.3][53.1, 69.0] [0.819, 1.11]  [105, 168] [103, 136] [0.795, 1.01] (p =0.440) (p = 0.061) AUC_(INF) 994 1480 1.48 1800 2800 1.56 (hr * IU/dL) [723, 1370] [1160, 1880] [1.26, 1.76] [1350, 2400] [1980, 3970] [1.33,1.83] (p = 0.002) (p < 0.001) t_(1/2) (hr) 12.2 18.8 1.54 11.0 1.8 1.70[9.14, 16.3] [14.8, 23.8] [1.40, 1.69] [8.76, 13.9] [14.3, 24.5] [1.54,1.89] (p < 0.001) (p < 0.001) MRT (hr) 17.5 27.0 1.54 15.8 27.0 1.71[13.1, 23.4] [21.3, 34.2] [1.40, 1.69] [12.6, 19.9] [20.6, 35.3] [1.54,1.89] (p < 0.001) (p < 0.001) CL 2.49 1.68 0.673 3.61 2.32 0.642(mL/hour/ [1.80, 3.45] [1.31, 2.15] [0.569, 0.796] [2.71, 4.83] [1.64,3.29] [0.547, 0.753] kg) (p = 0.002) (p < 0.001) V_(ss) 43.9 45.4 1.0457.4 62.8 1.09 (mL/kg) [39.3, 49.0] [39.3, 52.5] [0.947, 1.13]  [48.3,68.3] [55.2, 71.5] [0.976, 1.22] (p = 0.357) (p = 0.107) Incremental2.56 2.44 0.952 2.04 1.83 0.894 Recovery [2.36, 2.78] [2.12, 2.81][0.819, 1.11]  [1.61, 2.59] [1.59, 2.10] [0.795, 1.01] (IU/dL (p =0.444) (p = 0.060) per IU/kg) CI = Confidence Interval; Geom. Mean =Geometric Mean; OBS = observed. Estimated means, 95% CI for means, andmean differences were transformed to obtain estimated geometric means,95% CI for geometric means, and geometric mean ratios, respectively.

The t_(1/2), MRT, CL, and V_(ss) appeared to be independent of dose(TABLE 8). The geometric mean t_(1/2) of rFVIIIFc was 18.8 hours forboth the 25 IU/kg and 65 IU/kg dose groups. This represents a 1.54 and1.70-fold improvement over that of ADVATE® (12.2 hours and 11.0 hours)at equivalent doses (p<0.001), respectively (TABLE 8). The sameintra-subject improvement was observed in the MRT of rFVIIIFc (27.0hours for both dose groups) compared with ADVATE® (17.5 hours for the 25IU/kg and 15.8 hours for the 65 IU/kg) (p<0.001). Consistent withimprovement in the t_(1/2) and MRT was a corresponding 1.49 and1.56-fold reduction in intra-subject CL at doses of 25 IU/kg (p=0.002)and 65 IU/kg (p<0.001), respectively. There were no significantdifferences in V_(ss) and incremental recovery between ADVATE® andrFVIIIFc. Therefore, within each subject, rFVIIIFc demonstrated animproved PK profile compared with ADVATE®.

The improved PK profile of rFVIIIFc resulted in increased timepost-dosing to 1% FVIII activity which was 1.53 and 1.68-fold longerrespectively, than with ADVATE® at 25 IU/kg (p<0.001) and 65 IU/kg(p<0.001) (data not shown), suggesting a potentially longer therapeuticduration for rFVIIIFc. The favorable PK profile of rFVIIIFc relative toADVATE® was also demonstrated by FVIII activity measured in thechromogenic assay (TABLE 9), which was comparable to data derived fromaPTT assays. The estimation of exposure, i.e., C_(max) and AUC_(INF),was slightly higher, however, based on the chromogenic assay than on theone-stage (aPTT) clotting assay for both ADVATE® and rFVIIIFc.

TABLE 9 PK Parameters by Two-Stage (Chromogenic) Assay for rFVIIIFc andADVATE ® Per Dose Group Dose: 25 IU/kg (N = 6) Dose: 65 IU/kg (N = 9)ADVATE ® Geom. ADVATE ® rFVIIIFc Geom. Geom. rFVIIIFc Mean Ratio Geom.Geom. Mean Ratio Mean Geom. Mean [95% CI] (p- Mean Mean [95% CI]Parameter [95% CI] [95% CI] value) [95% CI] [95% CI] (p-value) C_(max)_(—) OBS 75.5 76.5 1.01 175 182 1.04 (IU/dL) [65.5, 87.1] [64.9, 90.1][0.940, 1.09]  [143, 215] [146, 227] [0.900, 1.20]  (p = 0.686) (p =0.571) AUC_(INF) 1060 1660 1.57 2270 4280 1.89 (hr * IU/dL) [822, 1360][1300, 2120] [1.38, 1.80] [1670, 3070] [2960, 6190] [1.61, 2.21] (p <0.001) (p < 0.001) t_(1/2) (hr) 10.5 16.7 1.59 10.8 19.8 1.84 [8.49,12.9] [13.8, 20.1] [1.35, 1.87] [8.16, 14.2] [14.3, 27.5] [1.60, 2.12](p < 0.001) (p < 0.001) MRT (hr) 15.0 23.9 1.59 15.4 28.5 1.85 [12.2,18.6] [19.8, 28.9] [1.35, 1.87] [11.7, 20.4] [20.5, 39.6] [1.61, 2.12](p < 0.001) (p < 0.001) CL 2.35 1.49 0.636 2.87 1.52 0.530 (mL/hour/[1.80, 3.06] [1.16, 1.92] [0.557, 0.727] [2.12, 3.89] [1.05, 2.20][0.453, 0.620] kg) (p < 0.001) (p < 0.001) Vss 35.5 35.9 1.01 44.5 43.40.975 (mL/kg) [30.5, 41.3] [30.4, 42.3] [0.898, 1.14] [36.7, 54.1][38.2, 49.4] [0.863, 1.10] (p = 0.822) (p = 0.653) Incremental 3.05 3.091.01 2.70 2.80 1.04 Recovery [2.62, 3.54] [2.61, 3.66] [0.940, 1.09][2.20, 3.31] [2.24, 3.50] [0.900, 1.20]  (IU/dL (p = 0.679) (p = 0.571)per IU/kg) CI = Confidence Interval; Geom. Mean = Geometric Mean; OBS =observed. Estimated means, 95% CI for means, and mean differences weretransformed to obtain estimated geometric means, 95% CI for geometricmeans, and geometric mean ratios, respectively.

Correlation Between von Willebrand Factor and Disposition of rFVIIIFc:

Because the majority of FVIII in circulation is in complex with VWF,Lenting P. J. et al., J Thromb Haemost. 5: 1353-60 (2007) and becausethe genome-wide association study has identified that the geneticdeterminants of FVIII levels are primarily dependent on VWF levels,Smith N. L. et al., Circulation 121:1382-1392 (2010) we examined theassociation between VWF and rFVIIIFc. A strong correlation was observedbetween VWF levels and CL and t_(1/2) for both rFVIIIFc and ADVATE®. Asshown in FIGS. 11A and 11B, as the level of VWF increased, the CL ofrFVIIIFc (p=0.0016) and of ADVATE® (p=0.0012) decreased.

The opposite relationship was observed between the level of VWF andt_(1/2). As the level of VWF increased, the t_(1/2) of rFVIIIFc(p=0.0003) and of ADVATE® (p<0.0001) increased. This correlationindicates that the Fc moiety of rFVIIIFc does not alter the role of VWFin protecting FVIII from clearance.

Effects of Prolonged PK of rFVIIIFc on Whole Blood ROTEM®:

Prior to administration of study drug, blood from one subject in eachdose group was spiked with an equal dose of rFVIIIFc or ADVATE® andanalyzed by whole blood ROTEM®. Clotting time (CT) was linear withrespect to the dose in the range of approximately 1% to 100% of normal,and the dose response was comparable between rFVIIIFc and ADVATE® in thesame subject (data not shown), indicating comparable potency of rFVIIIFcand ADVATE® in clot formation.

Despite the variable baseline CT due to residual FVIII levels prior tothe administration of ADVATE® or rFVIIIFc, both products effectivelycorrected the CT to comparable levels 30 minutes post-dosing (see FIGS.12A and 12B). The improvement in CT was better sustained by rFVIIIFcthan ADVATE® after 3 hours following a dose of 25 IU/kg (FIG. 12A), andafter 24 hours following a dose of 65 IU/kg (FIG. 12B).

rFVIIIFc was well tolerated by subjects at both doses. There were noclinically significant changes observed in hematology, blood chemistry,or urinalysis parameters. The majority of AEs were mild, unrelated torFVIIIFc, and resolved without sequelae. No serious AEs or deathsoccurred during the study, and no subjects at either dose developedneutralizing or binding antibodies to rFVIIIFc.

rFVIIIFc demonstrated a significantly improved FVIII activity PK profilerelative to ADVATE®, with t_(1/2) and MRT across dose levels being 1.54to 1.71-fold longer, as measured by the one-stage (aPTT) clotting assayand 1.59 to 1.84-fold longer by the two-stage chromogenic assay. Theprolonged activity of rFVIIIFc predicts possible prolonged efficacy,allowing for a less frequent dosing regimen in the prophylactictreatment of patients with Hemophilia A.

Adopting the PK parameters derived from this study, the Monte Carlosimulation predicted that a higher percentage of patients receivingrFVIIIFc will sustain FVIII levels above 1% or 3% as compared withpatients receiving equal doses of ADVATE® (TABLE 10). For example, at adose of 25 IU/kg, 12.2% of ADVATE® patients versus 71.2% of rFVIIIFcpatients were predicted to have FVIII trough levels above 1% on Day 4;at a dose of 65 IU/kg, 11.0% of ADVATE® patients versus 66.4% ofrFVIIIFc patients are predicted to have FVIII levels above 3% on Day 4.Clinical trials in larger numbers of patients are planned to confirmresults from this Phase 1/2a study and from the Monte Carlo simulationpredictions.

TABLE 10 Predicted Percentage of Subjects Achieving FVIII Trough LevelsAbove 1% and 3% of Normal at a Specified Dose Regimen of ADVATE ® orrFVIIIFc Timepoint following ADVATE ® rFVIIIFc dosing (Day) 25 IU/kg 65IU/kg 25 IU/kg 65 IU/kg Percent of Subjects with FVIII Trough Levelsabove 1% 3 40.0 67.8 92.6 99.0 4 12.2 31.0 71.2 90.0 5 4.20 13.6 39.471.6 7 0.200 1.40 7.80 26.4 Percent of Subjects with FVIII Trough Levelsabove 3% 3 10.6 34.6 62.2 91.0 4 1.60 11.0 25.4 66.4 5 0.200 3.20 7.0036.2 7 0 0.200 0.400 6.60

In vitro coagulation assays demonstrate no loss of specific activity forrFVIIIFc, compared to B-domain deleted or native FVIII, by eitherclotting or chromogenic assays, using commercially available reagentsand commonly used FVIII reference standards (Dumont et al., Blood(2012), prepublished online DOI:10.1182/blood-2011-08-367813). Inaddition, these results indicate that rFVIIIFc can be reliably assayedin a clinic setting by either the one-stage assay or the chromogenicmethod.

In summary, this Phase 1/2a clinical trial has demonstrated the safetyand prolonged t_(1/2) of rFVIIIFc in patients with severe hemophilia A.A pivotal Phase 3 study is ongoing with rFVIIIFc to establish effectiveprophylaxis dosing regimens for individuals with hemophilia A.

Example 10 Pharmacokinetics and Efficacy of rFVIIIFc in Mouse and DogModels of Hemophilia A

The pharmacokinetics and efficacy of rFVIIIFc compared to rFVIII wasevaluated in mouse and dog models of hemophilia A, in support of humanstudies. rFVIIIFc is a heterodimeric protein comprising a singleB-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain ofhuman immunoglobulin G1 (IgG1). Traditional dimeric Fc fusions, createdthrough the fusion of the monomeric effector protein to a monomer of Fcand then coupled through a disulfide bond to create a dimer, were noteffective for large coagulation proteins such as FVIII. Thus, we havedeveloped methods to create novel Fc fusion protein constructs in whicha single (monomeric) effector molecule is attached to Fc (Dumont J. A.,et al., BioDrugs 20(3):151-60 (2006)), (Dumont J. A., et al., Journal ofaerosol medicine. 18(3):294-303 (2005)), (Bitonti, et al., Proc NatlAcad Sci U.S.A. 101(26):9763-9768 (2004)). We have applied this approachto a number of proteins, including human rFIX (Peters, R. T., et al.,Blood. 115(10):2057-2064 (2010)), rFVIIa (Salas, J., et al., J ThrombHaemost. 9(s2):O-TU-026. doi: 10.1111/j.1538-7836.2011.04380_2.x(2011)), and BDD rFVIII.

Methods and Materials

Recombinant FVIII-Fc Fusion Protein (rFVIIIFc):

The rFVIIIFc expression plasmid pBUDCE4.1 (Invitrogen) contained twoexpression cassettes. One expressed, under the control of CMV promoter,native human FVIII signal sequence followed by BDD FVIII (S743 to Q1638fusion) directly linked to the Fc region of human IgG1 (amino acids D221to K457, EU numbering) with no intervening sequence. The other used theEF1α promoter to express the Fc region alone with a heterologous mouseIgκB signal sequence. Human embryonic kidney 293 cells (HEK293H,Invitrogen) were transfected with this plasmid, and a stable clonalsuspension cell line was generated that expressed rFVIIIFc. Protein waspurified from defined cell culture harvest media using a three columnpurification process, including a FVIII-specific affinity purificationstep (McCue J. et al., J. Chromatogr. A., 1216(45): 7824-30 (2009))followed by a combination of anion exchange and hydrophobic interactionchromatographic steps.

Recombinant FVIII (rFVIII):

Recombinant BDD FVIII (REFACTO® and XYNTHA®), and full length FVIII(ADVATE®) were purchased from Novis Pharmaceuticals (Miami, Fla.) andreconstituted according to the manufacturer's instructions.

Animals:

The hemophilia A (HemA) mice bearing a FVIII exon 16 knockout on a129×B6 background were obtained from Dr. H. Kazazian at the Universityof Pennsylvania (Bi, L., et al., Nat Genet. 10(1):119-121 (1995)) andbred at Biogen Idec Hemophilia. Murine FcRn knockout (FcRn KO) and humanFcRn transgenic (Tg32B) mice were derived from C57BL/6J mice and wereobtained from Dr. Derry Roopenian of The Jackson Laboratory in BarHarbor, Me. The genotypes for FcRn KO mice are mFcRn (−/−) and mβ2m(−/−), and for Tg32B are mFcRn (−/−), mβ2m (−/−), hFcRn (+/+), and hβ2m(+/+). C57BL/6 mice were purchased from The Jackson Laboratories (BarHarbor, Me.). All animal activities were approved by the InstitutionalAnimal Care Committees and performed in accordance with the “Guide tothe Care and Use of Laboratory Animals.”

Hemophilia A dogs were from the in-bred colony maintained at the FrancisOwen Blood Research Laboratory at the University of North Carolina,Chapel Hill. These dogs have a severe hemophilic phenotype comparable tothe severe form of the human disease (Graham, J. B. and Buckwalter, J.A., et al., The Journal of Exp. Med. 90(2):97-111 (1949)), (Lozier, J.N., et al., Proc Natl Acad Sci U.S.A. 99(20): 12991-12996 (2002)).

Pharmacokinetic (PK) Studies in Mice:

The PK of rFVIIIFc and rFVIII (XYNTHA®) was evaluated in HemA, C57BL/6,FcRn KO, and Tg32B mice after an intravenous dose of 125 IU/kg. Bloodwas collected from the vena cava in one-tenth volume of 4% sodiumcitrate at 5 minutes, and 4, 8, 16, 24, 32, 48, 54, and 72 hourspost-dosing for rFVIIIFc and at 5 minutes, and 1, 4, 8, 16, 20, 24, 32,and 48 hours post-dosing for rFVIII (4 mice/time point/treatment).Plasma was snap frozen in an ethanol/dry ice bath and stored at −80° C.until analysis for FVIII activity using a human FVIII-specificchromogenic assay (FVIII Coatest SP kit from DiaPharma [West Chester,Ohio]). The pharmacokinetic parameters were estimated bynon-compartmental modeling using WINNONLIN® version 5.2 (Pharsight,Mountain View, Calif.).

Efficacy Studies in HemA Mice:

All efficacy studies were performed blinded. Acute efficacy was studiedin the tail clip bleeding model. Male HemA mice (8-12 weeks old) wereanesthetized with a cocktail of 50 mg/kg of Ketamine and 0.5 mg/kg ofDexmedetomidine. The tail was then immersed in 37° C. saline for 10minutes to dilate the lateral vein followed by tail vein injection ofrFVIIIFc, rFVIII (ADVATE®), or vehicle. Five minutes later, the distal 1cm of the tail was clipped and the shed blood was collected into 13 mLof warm saline for 30 minutes. The blood loss was quantifiedgravimetrically.

The prophylactic efficacy was studied in the tail vein transection (TVT)bleeding model as described previously (Pan, J. and Kim, J. Y., Blood.114(13):2802-2811 (2009)) except that HemA mice received a singleintravenous administration of 12 IU/kg of rFVIIIFc, rFVIII (ADVATE®), orvehicle at 24 or 48 hours prior to the transection of a lateral tailvein. The dose of 12 IU/kg was identified from a prior dose responseexperiment with rFVIII in which 12 IU/kg achieved 50% protection of HemAmice from a TVT injury inflicted 24 hours post dosing (data not shown).

Hemophilia A Dog Studies:

In a single dose PK/PD study of rFVIIIFc, two naïve hemophilia A dogs(M10 and M11) received an intravenous dose of 125 IU/kg. Blood sampleswere collected pre-dosing and post-dosing at 5 and 30 min, and 1, 2, 4,8, 24, 32, 48, 72, 96, 144, and 168 hours for whole blood clotting time(WBCT). Blood collections for FVIII activity (aPTT and chromogenicassay), rFVIIIFc antigen (ELISA), hematology, and blood chemistryincluded the time points listed above for WBCT as well as 15 minute and3, 6, and 12 hours post-dosing.

In the following sequential design study, rFVIII (REFACTO®) wasadministered intravenously at 114 IU/kg for dog M12 and 120 IU/kg fordog M38. WBCT was measured until clotting times were ≥20 minutes (thetime consistent with FVIII:C≤1%), and samples were also collected at thespecified time points for FVIII activity (aPTT and chromogenic assay),antigen (ELISA). and hematology tests. Then 125 IU/kg rFVIIIFc wasadministered intravenously to the same dogs and blood samples werecollected for WBCT, aPTT, ELISA, hematology, and serum chemistry. Timepoints for WBCT included pre-dosing, and 5 and 30 minutes and 1, 2, 4,8, 24, 32, 48, and 72 hours post-dosing of rFVIII and rFVIIIFc. Bloodwas also collected at 96, 120, 144, and 168 hours post-dosing withFVIIIFc. Blood collections for FVIII activity and antigens included thetime points listed above for WBCT as well as 15 minutes and 3, 6, 12hours after dosing. The WBCT and aPTT were performed as previouslydescribed (Herzog, et al., Nat Med. 5(1):56-63 (1999)).

FVIII Chromogenic Assays:

FVIII activity in hemophilia A dog plasma was tested by an automatedchromogenic assay on a Sysmex CA1500 instrument (Sysmex, IL) withreagents from Siemens Healthcare Diagnostics (Dallas, Tex.). Thestandard curve was generated with the 7th International Standard FVIIIConcentrate (NIBSC code 99/678) spiked into human FVIII-depleted plasma(Stago, USA) at concentrations ranging from 1.5-0.016 IU/mL.

FVIII activity in HemA mouse plasma was measured using the Coatest SPFVIII assay from Chromogenix (DiaPharma, Lexington, Mass.), followingthe manufacturer's instructions. The standard curve was generated usingrFVIIIFc or rFVIII serially diluted from 100 mU/mL to 0.78 mU/mL inbuffer containing naive HemA mouse plasma. To measure the human FVIIIactivity in C57BL/6, FcRn KO, and Tg32B mouse plasma, the infusedrFVIIIFc or rFVIII in mouse plasma was first captured by humanFVIII-specific mAb GMA8016 (Green Mountain Antibodies, VT) followed bythe standard Coatest assay.

rFVIII- and rFVIIIFc-Specific ELISA:

rFVIII and rFVIIIFc antigen levels in hemophilia A dog plasma weremeasured by ELISA following the standard protocol. The FVIII A1domain-specific mAb GMA-8002 (Green Mountain Antibodies, Burlington,Vt.) was used as the capture antibody. HRP-conjugated polyclonalanti-FVIII Ab F8C-EIA-D (Affinity Biologicals) was used to detectrFVIII. HRP-conjugated donkey anti-human (F(ab)′₂) 709-036-098 (JacksonImmunologicals) was used to detect rFVIIIFc.

SPR Analysis of rFVIIIFc-FcRn Interactions:

Surface plasmon resonance (SPR) experiments were performed with aBiacore T100 instrument. Research-grade CM5 sensor chips, buffers, andimmobilization reagents were purchased from Biacore (GE Heathcare,Piscataway, N.J.). Single-chain human, canine, and murine FcRnpreparations were immobilized using standard amine coupling on adjacentflow cells of a single chip at a density of approximately 370 resonanceunits (RU), followed by blocking with ethanolamine. The steady-stateassociation of Fc-containing analytes (FVIIIFc and IgG) with immobilizedFcRn of different species was evaluated by sequential injection ofanalytes at 16 concentrations (0.0625-2000 nM) in pH 6.0 running buffer(50 mM MES [4-morpholineethanesulfonic acid], 250 mM sodium chloride, 2mM calcium chloride, 0.01% Tween 20 [polyethylene glycol sorbitanmonolaurate]). Each cycle was performed in duplicate and comprised a 45minutes association phase and a 15 minutes dissociation phase, both at aflow rate of 5 μL/min, followed by regeneration with two 60 secinjections of 1M Tris-HCl at 25 μL/min. After doublereference-subtraction (blank flow cell and running buffer alone),binding responses recorded near the end of the association phase wereplotted as a function of analyte concentration, and EC₅₀ values (50% ofR_(max)) were derived by non-linear regression analysis.

Statistical Analyses:

Unpaired t-test, one-way ANOVA, Mann-Whitney test, Kruskal-Wallis testwith Dunn post-test, survival curves and associated log-rank test wereperformed in GraphPad Prism 5 (Graph-Pad Software Inc., La Jolla,Calif.). A 2-tailed P value less than 0.05 was considered statisticallysignificant.

Results

Recombinant FVIII Fc Fusion Protein (rFVIIIFc):

rFVIIIFc is a recombinant fusion of human B-domain deleted FVIII with Fcfrom human IgG1, with no intervening linker sequence (FIG. 14), that wasproduced in well characterized HEK 293H cells. The rFVIIIFc isproteolytically cleaved intracellularly to yield an ˜90 kDa heavy chainand ˜130 kDa light chain-Fc that are bound together non-covalentlythrough a metal bond interaction mediated by the A1 and A3 domains ofFVIII.

The average specific activity of rFVIIIFc from fourteen separate batcheswas 8460±699 IU/mg by the one stage clotting (aPTT) assay, and 9348±1353IU/mg by the chromogenic assay, corresponding to 1861±154 and 2057±298IU/nmol, respectively. The specific activity of rFVIIIFc is comparableto that of wild type human FVIII in plasma (1429 IU/nmol) (Butenas, S.and Mann, K. G., Biochemistry (Mosc). 67(1):3-12 (2002)). Thus the FVIIIactivity of rFVIIIFc is not affected by fusion of the C-terminus ofhuman FVIII to the N-terminus of human Fc, and the results obtained withthe aPTT and chromogenic assays are within approximately 10% of oneanother.

Binding of rFVIIIFc to FcRn:

The affinity of rFVIIIFc for single-chain mouse, canine, and human FcRnwas evaluated using surface plasmon resonance. The rates of associationand dissociation for the complex between rFVIIIFc and mouse FcRn weremuch slower than those for canine and human FcRn. Half-maximal binding(EC₅₀) of rFVIIIFc to human FcRn was approximately 4-fold weaker thanthat to canine FcRn, and more than 20-fold weaker than that to mouseFcRn (TABLE 11). Similarly, human IgG1 also showed the highest affinityto murine FcRn, while binding affinity to canine FcRn was less comparedto murine FcRn, but greater compared to human FcRn (TABLE 11).

TABLE 11 Surface Plasmon resonance analysis of murine, canine, and humanFcRn with rFVIIIFc and human IgG1 Fc Sample * FcRn FcRn Density (RU)EC₅₀ (nM) ^(†) Rmax (RU) rFVIIIFc murine 370   <1.5 ^(‡) 581.4 rFVIIIFccanine 367  8.6 499.3 rFVIIIFc human 369  33.4 365.4 Human IgG1 murine378   <22.4 ^(‡) 320.0 Human IgG1 canine 367 196.3 282.2 Human IgG1human 378 558.4 211.0 * rFVIIIFc or IgG1 were injected over a flow cellto which various FcRn molecules were chemically conjugated atapproximately equal densities (~370 RU). ^(†) EC₅₀ values (50% ofR_(max)) were the average derived from non-linear regression analysis ofthe binding response curves fitted to 16 analyte concentrations(0.0625-2000 nM) repeated in duplicate. ^(‡) Due to the high affinities,the low binding curves at low analyte concentrations did not reachequilibrium under the normal operating conditions of the instrument.

FcRn-Dependent Improvement in Pharmacokinetics of rFVIIIFc in Mice:

Interaction of Fc with FcRn is considered the underlying mechanism forextending half-life for IgG and Fc-fusion proteins. To confirm that thismechanism of action is also responsible for extending half-life ofrFVIIIFc, we compared pharmacokinetic (PK) profiles of rFVIIIFc withrFVIII in FVIII-deficient (HemA) mice (FIG. 15A), normal (C57BL/6) mice(FIG. 15B), FcRn-deficient (FcRn KO) mice (FIG. 15C), and human FcRntransgenic (Tg32B) mice (FIG. 15D) following a single intravenousadministration of 125 IU/kg.

The PK parameters (TABLE 12) were determined by the chromogenicmeasurement of the human FVIII activity in mouse plasma. The t_(1/2) ofrFVIIIFc was 1.8-2.2 fold longer than rFVIII in HemA mice (13.7 vs 7.6hours) and normal mice (9.6 vs 4.3 hours). The t_(1/2) extension ofrFVIIIFc relative to rFVIII was abolished in FcRn KO mice (6.4 vs 6.9hours) and restored in human FcRn transgenic Tg32B mice (9.6 vs 4.1hours). The results thus confirm that the interaction of rFVIIIFc withthe FcRn receptor is responsible for its extended t_(1/2). Furthermore,consistent with the improved t_(1/2), rFVIIIFc also showed a 1.6-2.4fold longer MRT and a 1.2-1.8 fold increased systemic exposure (AUC)compared to rFVIII in FcRn-expressing (HemA, C57Bl/6 and Tg32B) mice butnot in FcRn KO mice.

TABLE 12 Summary of PK parameters for rFVIIIFc and rFVIII in differentmouse strains hFcRn Transgenic HemA^(†) C57BL/6^(†) FcRn KO^(†)(Tg32B)^(†) Mouse Strain rFVIIIFc* rFVIII* rFVIIIFc* rFVIII* rFVIIIFc*rFVIII* rFVIIIFc* rFVIII* C_(max) 2613.6 2710.4 2356.2 2000.1 2734.92458.4 3135.3 3137.0 (mIU/mL) Half-life 13.7 7.6 9.6 4.3 6.4 6.9 9.6 4.1(hr) MRT 17.6 11.0 9.8 5.4 6.3 8.5 12.8 5.4 (hr) Vss 68.2 49.2 67.5 50.849.3 51.6 64.1 49.6 (mL/kg) CL 3.9 4.5 6.9 9.3 7.8 6.1 4.1 7.3(mL/hr/kg) AUC 32332.4 28026.8 18089.1 13404.0 16087.2 20609.3 30534.517165.7 (hr * mIU/mL) C_(max): maximum plasma FVIII activity postinfusion; MRT: mean residence time; Vss: volume of distribution atsteady-state; ‘CL: clearance;’ AUC: area under the curve. ^(†)PKparameters of rFVIII and rFVIIIFc were compared only within the samemouse strain not across strains, because the same molecule can displaydifferent t_(1/2) in different mouse strains. *The PK evaluation of eachmolecule used a cohort of 36 mice, sampled by terminal vena cavableeding from 4 mice at each of the 9 time points. The group means ateach time point were used for non-compartment modeling in WINNONLIN ® toderive PK parameter estimates.

rFVIIIFc is Fully Active in Treating Acute Bleeds in HemA Mice:

To evaluate the acute efficacy of rFVIIIFc in comparison to rFVIII, HemAmice (16-20 mice/group) were treated with escalating doses (24, 72, and216 IU/kg) of rFVIIIFc or rFVIII and injured by tail clip 5 minutespost-dosing. In comparison to vehicle-treated mice (n=18) that had amedian blood loss of 1 mL, both rFVIIIFc and rFVIII treatments resultedin significantly improved protection (P<0.05, Kruskal-Wallis test withDunn post-test) (FIG. 16). The median blood loss progressively decreasedwith increasing doses, reaching maximum reduction to 0.23 mL at 72 IU/kgof rFVIIIFc, and 0.20 mL at 216 IU/kg of rFVIII. Overall, the blood losswas comparable in animals treated with equal doses of rFVIIIFc orrFVIII, indicating that both therapeutics are comparably active inresolving acute arterial bleeds.

Prolonged Prophylactic Efficacy of rFVIIIFc in HemA Mice:

To determine if prolonged PK lead to prolonged protection from injury,we compared the prophylactic efficacy of rFVIIIFc and rFVIII in HemAmice. Twenty-four hours after an intravenous dose of 12 IU/kg, onelateral tail vein in HemA mice was transected. Following injury, 49% ofrFVIII-treated mice (n=39) survived, compared with 100% survival ofrFVIIIFc-treated mice (n=19) (P<0.001, Log-Rank test) (FIG. 17A). Tofurther demonstrate that rFVIIIFc sustains a longer duration ofefficacy, HemA mice were injured 48 hours post-dosing with 12 IU/kg ofrFVIIIFc. Nevertheless, 58% of rFVIIIFc-treated mice (n=40) survived,which is similar to that achieved in rFVIII-treated mice (49%) injuredat 24 hours post dosing (FIG. 17A). Both rFVIIIFc and rFVIII treatmentsare significantly better than the HemA vehicle-control group (n=30) inwhich only 3% of mice survived the injury (P<0.0001) (FIG. 17A). Theimproved and prolonged prophylactic efficacy of rFVIIIFc is also evidentby the measurement of rebleeding post injury (FIG. 17B). Whereas 100% ofvehicle-treated HemA mice rebled within 10 hours following tail veintransection, 87% of rFVIII-treated and 47% of rFVIIIFc-treated micerebled after the injury inflicted at 24 hours post dosing, respectively(P=0.002, rFVIIIFc vs rFVIII) (FIG. 17B). The rebleed profile forrFVIIIFc-treated mice injured at 48 hours is largely comparable to thatfor rFVIII-treated mice injured at 24 hours post dosing. In contrast,both the survival and rebleed profile for rFVIII-treated mice injured at48 hours are indistinguishable from the profile for the vehicle-controlgroup (data not shown). Therefore, the results indicate that rFVIIIFcprotects HemA mice from tail vein injury twice as long as that achievedby the same dose of rFVIII.

Improved PK/PD of rFVIIIFc in Hemophilia A Dogs:

The PK and pharmacodynamics (PD) of rFVIIIFc were also studied inhemophilia A dogs. Following an intravenous dose of 125 IU/kg ofrFVIIIFc, the WBCT was immediately corrected to normal, which is in therange of 8-12 minutes in normal dogs (FIGS. 18A and B). The WBCTremained below 20 min, indicating FVIII activity >1%, throughapproximately 4 days in 3 out of 4 rFVIIIFc-treated dogs and 3 days inthe remaining dog (FIG. 18A). In dog M12 treated with 114 IU/kg ofrFVIII and dog M38 with 120 IU/kg of rFVIII, the WBCT was also correctedto normal immediately after dosing. However, the WBCT remained below 20minutes for 2 days in M12 and 3 days in M38, approximately 1.5-2-foldshorter than that achieved by rFVIIIFc (FIG. 18B). Furthermore, bothrFVIIIFc and rFVIII treatment also improved aPTT clotting time similarlyat 5 minutes post dosing.

The PK of rFVIIIFc antigen (FIG. 19A) was determined by measuring theconcentration of rFVIIIFc in plasma with a rFVIIIFc-specific ELISA thatdetects both the FVIII and Fc portions of the molecule. The t_(1/2) ofrFVIIIFc antigen is 15.7±1.7 hr (FIG. 19A), similar to the t_(1/2) ofrFVIIIFc activity (FIG. 19B), as measured by the chromogenic assay:15.4±0.3 hr (TABLE 13). There is a good correlation between the FVIIIactivity and the rFVIIIFc antigen data, thereby demonstrating thatrFVIIIFc protein was fully active in vivo.

TABLE 13 Summary of PK parameters for rFVIIIFc and rFVIII in hemophiliaA dogs PK by FVIII activity measurement C_(max) AUC T_(1/2) CL VssTreatment (IU/mL) (hr · IU/mL) (hr) (mL/hr/kg) (mL/kg) rFVIIIFc* 2.0 ±0.54 25.9 ± 6.47 15.4 ± 0.3 5.1 ± 1.4 86.4 ± 14.0 rFVIII^(†) 2.0 18.27.4 6.5 64.0 PK by rFVIII and rFVIIIFc antigen measurement C_(max) AUCT_(1/2) CL Vss Treatment (ng/mL) (hr · ng/mL (hr) (mL/hr/kg) (mL/kg)rFVIIIFc* 210 ± 33 2481 ± 970 15.7 ± 1.7 6.2 ± 3.0 86.1 ± 19.2rFVIII^(†) 211 1545 6.9 8.7 80.7 *Results presented are Mean ± SD from 4dogs. ^(†)Results presented are Mean. SD not reported since two dogswere utilized.

In two of the dogs (M12 and M38) that also received a single dose ofrFVIII 72 hours prior to dosing with rFVIIIFc, the t_(1/2) of rFVIIIantigen was determined to be 6.9 hours and rFVIII activity 7.4 hours.Therefore, the plasma half-life of rFVIIIFc was approximately twice aslong compared to that for rFVIII by both antigen and activitymeasurements.

In addition, platelet count and fibrinogen were assessed to serve aspreliminary tests for thrombogenicity. After dosing with either rFVIIIFcor rFVIII, platelet numbers and plasma fibrinogen concentration did notchange from pre-dose values (data not shown).

Discussion

These studies have shown rFVIIIFc to be fully active in treating acutebleeds in HemA mice, in addition to retaining normal specific activity.Other studies, not reported here, have shown that rFVIIIFc is also fullyfunctional in interacting with FIXa, FX, and phospholipids in formingthe Xase complex (Peters et al., J. Thromb Haemost. DOI:10.1111/jth.12076 (2012)). Furthermore, the binding affinity to vonWillebrand Factor (VWF) was comparable between rFVIIIFc and rFVIII, witha Kd of approximately 1.4 and 0.8 nM for rFVIIIFc and rFVIII,respectively (Peters et al., J. Thromb Haemost. DOI: 10.1111/jth.12076(2012)).

The activity of rFVIIIFc was not affected by fusion of the C-terminus ofFVIII with the N-terminus of Fc since the C1 and C2 domains of FVIIIwere involved in phospholipid binding which is essential for theformation of prothrombinase complex on activated platelet surfaces(Foster, P. A., et al., Blood. 75(10):1999-2004 (1990)). However, thisfinding was consistent with the observation that residues thought tobind phospholipids, e.g., K2092/F2093 in C1, M2199/F2200 and L2251/L2252in C2, all appear to form a surface that is distant from the C-terminalresidues of FVIII (Shen, B. W., et al., Blood. (2007); Ngo, J. C., etal., Structure. 16(4):597-606 (2008)).

The half-life of rFVIIIFc was doubled only in mice expressing eitherendogenous murine or transgenic human FcRn, but not in FcRn KO mice (seeFIG. 15 and TABLE 12), demonstrating that the mechanism of prolonginghalf-life of rFVIIIFc is mediated by FcRn. While it is known that bothendothelial and hematopoietic cells contribute equally in recyclinginternalized IgG to the cell surface to facilitate the longevity of IgGand protection from degradation, (Borvak, J., et al., Int Immunol.10(9):1289-1298 (1998)), (Akilesh, S., et al., J Immunol.179(7):4580-4588 (2007)) it is not known specifically whichFcRn-expressing cell type(s) are responsible for the uptake andrecycling of rFVIIIFc. FcRn is broadly expressed in the vascularendothelium, epithelium of kidney, liver, spleen, as well as in bonemarrow-derived APCs including macrophages (Borvak, J., et al., IntImmunol. 10(9):1289-1298 (1998)), (Akilesh, S., et al., J Immunol.179(7):4580-4588 (2007)), (Yoshida, M., et al., Immunity. 20(6):769-783(2004)). Since FVIII circulates largely (˜98%) in complex with VWF(Lenting, P. J., et al., J Thromb Haemost. 5(7):1353-1360 (2007)), andboth proteins were colocalized to the macrophages in liver and spleenwhen recombinant FVIII and VWF were co-injected into VWF-deficient mice(van Schooten, C. J., et al., Blood. 112(5):1704-1712 (2008)),macrophages can play a role in rescue of rFVIIIFc from degradation andprolongation of half-life. However, the results can also be indicativeof a previously unrecognized pathway for FVIII catabolism, and rescue ofthe protein permitted by Fc fusion.

Approaches in development to extend the half-life of clotting factorsinclude pegylation (Rostin, J., et al., Bioconjug Chem. 11(3):387-396(2000)), (Mei, B., et al., Blood. 116(2):270-279 (2010)),glycopegylation (Moss, J., et al., J Thromb Haemost. 9(7):1368-1374(2011)), (Negrier, C., et al., Blood. (2011)), and conjugation withalbumin (Metzner, H. J., et al., Thromb Haemost. 102(4):634-644 (2009)),(Weimer, T., et al., Thromb Haemost. 99(4):659-667 (2008)). Regardlessof the protein engineering utilized, the half-life of modified rFVIIIvariants appears to be maximally twice as long as wild-type FVIII in avariety of preclinical animal models (Liu, T., et al., Blood. 112:511(2008)), (Karpf, D. M., et al., 16(Suppl. S4):40 (2010)). Consistentresults have been demonstrated in humans, e.g., rFVIIIFc was reported toimprove half-life approximately 1.7-fold compared to ADVATE® inhemophilia A patients (Powell, J. S., et al., Blood. (2012) prepublishedonline. DOI:10.1182/blood-2011-09-382846). This limitation of extendingFVIII half-life appears to be related to VWF. In FVIII and VWF knockoutmice, preliminary experiments observed a 5-fold increase in thehalf-life of rFVIIIFc compared to rFVIII (Liu, T et al., unpublishedresults). Similar findings were reported previously in VWF knockout miceutilizing Pegylated-FVIII (Mei, B., et al., Blood. 116(2):270-279(2010)). Taken together, these results indicate that VWF can be alimiting factor for further extending FVIII half-life.

Beyond extending half-life, rFVIIIFc provides additional benefits. Onemajor challenge with FVIII replacement therapy is the development ofneutralizing anti-FVIII antibodies (inhibitors). This occurs in 15-30%of previously untreated patients. rFVIIIFc has the potential to induceimmune tolerance and thus prevent the development of neutralizingantibodies. It has been reported that retroviral vector-transducedB-cells, presenting FVIII domains as Ig fusion proteins, specificallyprevent or decrease existing FVIII antibodies in HemA mice (Lei, T. C.and Scott, D. W., Blood. 105(12):4865-4870 (2005)). It was also foundthat Fc contains regulatory T-cell epitopes capable of inducing Tregexpansion and suppression of antigen-specific immune responses in vitro(De Groot, et al., Blood. 112(8):3303-3311 (2008)). In addition, theFcRn-mediated transfer of maternal IgG and Fc-fusion proteins acrossplacenta to fetal circulation (Simister, N. E., Vaccine.21(24):3365-3369 (2003)), Grubb, J. H., et al., Proc Natl Acad SciU.S.A. 105(24):8375-8380 (2008)) could induce neonatal tolerance torFVIIIFc while also providing needed protection in the newborn frombleeding during delivery.

In conclusion, we have demonstrated that rFVIIIFc, providesapproximately 2-fold longer efficacy duration relative to rFVIII inprotecting HemA mice from tail vein transection injury and improvingWBCT in HemA dogs. The prolonged efficacy correlates well with a 2-foldextended t_(1/2) of rFVIIIFc, a result of recycling of the Fc fusionprotein via a specific and well established intracellular pathway.

Example 11 Immunogenicity of rFVIIIFc in Mice

130 male Hemophilia A mice, age 7-9 weeks old at the beginning of thestudy, were randomized into 13 treatment groups based on age and Bodyweight (n=10/group). Mice were treated with repeated intravenous dosingof either rFVIIIFc, rFVIII-mFc, XYNTHA® or ADVATE® at 50, 100 and 250IU/kg, a mixture of the three formulation buffers for FVIIIFc, XYNTHA®and ADVATE® was used for the vehicle control group. The IVadministration times were day 0, day 7, day 14, day 21, day 35 post thefirst IV injection and blood samples were collected via Retro-orbitalblood collection at day (−1), day 14, day 21, day 28 and day 42 post thefirst treatment (FIG. 20).

Immediately after blood collection, plasma samples were isolated throughcentrifugation and inactivated by 30 minute heat treatment at 56° C. toinsure the accurate measurement of anti-FVIII antibodies. Thedevelopment of total anti-FVIII antibody (FIGS. 21A, 21B and 21C),anti-FVIII neutralizing antibody (FIG. 23) and total anti-Fc antibody(FIG. 24) were investigated using the plasma samples.

When treated with 50 IU/kg FVIII, 28 days post the first injection, only1 out of 10 mice in the rFVIIIFc treatment group, 2 out of 10 mice inthe rFVIII-mFc treatment group developed detectable anti-FVIII antibodycompared to 5 out of 10 mice and 7 out of 10 mice for the XYNTHA® andADVATE® treated mice (FIG. 22C). At 100 IU/kg, the numbers of mice thathad detectable anti-FVIII antibody at day 28 were 2, 5, 8 and 9 in therFVIIIFc, rFVIII-mFc, XYNTHA® and ADVATE® treatment group respectively(FIG. 22C). At 250 IU/kg, a supra-physiological dose, the numbers ofmice that had detectable anti-FVIII antibody at day 28 were 10, 10, 7and 7 in the rFVIIIFc, rFVIII-mFc, XYNTHA® and ADVATE® treatment grouprespectively (FIG. 22C). Data corresponding to day 14, day 21, and day42 is shown in FIGS. 22A, 22B, and 22D, respectively.

In general, we observed a good correlation between the total andneutralizing antibodies to FVIII (R²=0.7452), and the titers of bothincreased over time (FIG. 23). Within the therapeutic dose range (50 and100 IU/kg), the number of mice that developed FVIII-specific antibodiesas well as the antibody titers in rFVIIIFc treatment groups weresignificantly lower compared to ADVATE® (p<0.05), and marginally lowerversus XYNTHA® (p=0.05). The results indicate a potentially lowimmunogenicity of rFVIIIFc.

Example 12 Splenic Lymphocyte Response to rFVIIIFc Compared withCommercially Available rFVIII

The splenic lymphocyte response to recombinant Factor VIII (rFVIII) whenlinked to either human Fc (hFc, IgG1) or mouse Fc (mFc, IgG2a) wasdetermined and compared with the splenic lymphocyte response tocommercially available rFVIII [full-length FVIII (ADVATE®) andB-domain-deleted FVIII (XYNTHA®/REFACTO AF®)].

HemA mice were injected once weekly for six weeks with either 50 or 250IU/kg of the tested molecules. On day 56, four mice from each group wereeuthanized and splenocytes were isolated (FIG. 25). One half of thesplenocytes was used for determining the splenic immunogenicity profileby staining for intracellular cytokines, markers for regulatory T cells,and dendritic cells using flow cytometry (FACS) (FIG. 26). The otherhalf of the cells were used for isolating RNA and carrying out pathwayprofiling using real time PCR based arrays. FIG. 27A shows a FACS dotplot profile of the isotype control. FIG. 27B shows a FACS dot plotprofile of a sample containing splenocytes positive for both CD4 andTNF-α. Percentage of double positive cells were determined from dotplots from all the treatments and vehicle. Percent of double positivecells in FVIII treated mice was obtained by comparing with vehicletreated group.

Intracellular cytokine staining was performed by co-staining for theT-helper cell marker CD4 and cytokines such as IL-2 (FIG. 28), IL-4(FIG. 30), and TNF-α (FIG. 29). IL-2 is a T-cell mitogen involved inT-cell proliferation, which is secreted by activated T-cells in responseto FVIII in hemA mice. IL-4 has been identified as a cytokine secretedby activated T cells in response to FVIII in hemA mice. TNF-α is apro-inflammatory cytokine responsible for higher antibody production inhemophilia patients. The fluorescence intensities of each of thestaining were measured using flow cytometry.

Similarly, the proportion of tolerogenic and immunogenic dendritic cellswas determined by surface staining and flow cytometry analysis ofmarkers such as PD-L1 (CD274) (FIG. 32) and CD80 (FIG. 33). PD-L1 is aninhibitory ligand that engages the PD-1 receptor on activated T-cellsthereby blocking T-cell Receptor (TCR) mediated production of IL-2 andproliferation. Higher DC expression of PD-L1 is a critical factor thatcan inhibit immunogenicity and promote tolerance. CD80 is a surfacemarker usually seen on dendritic cells upon phagocytosing antigen andduring maturation to present antigen to T cells. CD80 belongs to a panelof co-receptors in activating T cells for proliferation. More CD80surface staining indirectly indicates better maturation and antigenpresentation by dendritic cells.

In addition, the percentage of regulatory cells (Treg) in the spleen wasassessed by co-staining for CD4 and foxp3 (FIG. 34), a marker for thesecells. Foxp3 is an intracellular marker of regulatory T cells. Foxp3+T-cells are involved in the establishment, maintenance, and adoptivetransfer of T-cell mediated peripheral tolerance.

The presence of cells expressing both CD4 and the Tim3 (FIG. 35) orCD279 (PD-1) (FIG. 36) makers was also assessed. Tim3 (T-cellimmunoglobulin domain and mucin domain 3) as a negative regulator of Thelper 1 (Th1)-cell responses. Tim3 is also expressed by innate immunecells and can promote a pro-inflammatory response. Tim3 inhibitsTh1-mediated auto- and alloimmune responses and acts via its ligand,galectin-9, to induce cell death in Th1 but not Th2 cells. CD279 (PD-1)is a member of the extended CD28/CTLA-4 family of T cell regulators.PD-1 is expressed on the surface of activated T cells, B cells, andmacrophages suggesting that compared to CTLA-4, PD-1 more broadlynegatively regulates immune responses.

Among the tested cytokines that are responsible for immunogenicity andinhibitor formation, in mice injected with the 50 IU/kg of rFVIII-hFc orrFVIII-mFc, there was a significant inhibition in the levels of IL-4 andTNF-α and the levels of IL-2 did not change compared to vehicle injectedgroup. Conversely, the levels of these cytokines were higher in groupsreceiving 250 IU/kg of these molecules. Mice injected with 50 IU/kg ofeither XYNTHA® or ADVATE® did not exhibit any inhibition, whereas the250 IU/kg group showed an increase in intracellular content of IL-2,IL-4, and TNF-α. In addition, there was a higher percentage of foxp3positive T cells in mice injected with 50 IU/kg of rFVIII-mFc comparedto other treatments. Mice receiving 50 IU/kg of rFVIII-hFc andrFVIII-mFc had a higher percentage of splenic dendritic cells positivefor PD-L1 (CD274), an inhibitory signal for T-cell activation andproliferation. These groups also had a higher percentage of immaturedendritic cells as illustrated by a decrease in CD80 staining.

These results indicated that rFVIIIFc at 50 IU/kg in hemA mice exhibitedlower immunogenicity than commercially available rFVIII [full-lengthFVIII (ADVATE®) and B-domain-deleted FVIII (XYNTHA®/REFACTO AF®)].Accordingly, rFVIIIFc can promote lower antibody production and induceimmune tolerance.

Example 13 Biodistribution and Clearance of rFVIIIFc in Mice

Recombinant fusion of a single FVIII molecule to the constant region ofIgG1 Fc (rFVIIIFc) has been shown to decrease clearance compared torFVIII in an FcRn dependent manner (Powell et al., 2012 Blood), using anatural pathway that recirculates antibodies into the blood stream. Inaddition, as disclosed above, a phase 1/2a clinical trial in hemophiliaA subjects demonstrated that rFVIIIFc has a 1.5 to 1.7-fold longerhalf-life than recombinant full length FVIII (ADVATE®). Accordingly, astudy was conducted (i) to identify the cell types and organs thatcontribute to the protection of rFVIIIFc and (ii) to assess the relativecontributions of the FVIII and Fc domains to the biodistribution andclearance of rFVIIIFc in mice.

The clearance of rFVIIIFc to rFVIII was compared in geneticallyengineered KO mouse models deficient in either FVIII (HemA) or vonWillebrand Factor (VWF). Intravenously dosed clodronate-containing lipidvesicles were used to deplete Kupffer cells and monocytes/macrophages inthese mouse models. The effectiveness of depletion was quantitated byimmunohistochemistry and FACS analysis. Pharmacokinetic analysis wasperformed with a FVIII-specific Coatest assay following intravenousinjection of rFVIIIFc or rFVIII.

Kupffer cell depletion in HemA mice increased rFVIIIFc clearance.Furthermore, in the absence of VWF (HemA/VWF double knockout mice), thedepletion of Kupffer cells and macrophages increased the clearance ofrFVIIIFc to levels similar to that of rFVIII, indicating that thesecells are responsible for the majority of the difference in clearancebetween rFVIII and rFVIIIFc in this model.

These studies suggest that Kupffer cells can contribute to theFcRn-mediated recycling of rFVIIIFc. Studies using bone marrowtransplants with FcRn KO mice are in progress to verify this mechanism.These studies, combined with in vitro cellular uptake experiments willattempt to distinguish the contribution of Kupffer cells from otherFcRn-expressing cell types, including endothelial cells.

Example 14 Cell-Mediated Immune Response to rFVIIIFc in Hemophilia AMice

The objective of the present study was to identify cell-mediated immuneresponses to recombinant FVIII, which is of interest in designing bettertherapeutic management of hemophilia A. Thus, we investigated thesplenic lymphocyte response to recombinant FVIII (rFVIII) when linked tohuman Fc (rFVIIIFc; IgG1) in comparison with commercially availablefull-length rFVIII (ADVATE®) and B-domain-deleted rFVIII(XYNTHA®/REFACTO AF®). HemA mice were injected with 4 weekly followed by2 every other week doses of 50 100 or 250 IU/kg. At the end of 8 weeks,mice from each group were euthanized and their splenic leukocyteimmunogenicity profile was determined by testing for intracellularcytokines, markers for regulatory T cells and dendritic cells using flowcytometry and RNA profiling. In mice injected with the 50 IU/kg ofrFVIIIFc, there was a significant inhibition in the levels of IL-2, IL-4and TNF-α (cytokines that promote immunogenicity). The levels of thesecytokines were higher in mice receiving 250 IU/kg of this molecule. Miceinjected with 50 IU/kg of either XYNTHA® or ADVATE® did not exhibit anyinhibition, whereas the 250 IU/kg group showed an increase inintracellular content of IL-2, IL-4, and TNF-α. In addition, there was ahigher percentage of foxp3 positive T cells in mice injected with 50 and100 IU/kg of rFVIIIFc compared to other treatments. Mice receiving 50and 100 IU/kg of rFVIIIFc had a higher percentage of splenic dendriticcells positive for PD-L1 (CD279), an inhibitory signal for T-cellactivation and proliferation. These groups also had a higher percentageof immature dendritic cells as illustrated by a decrease in CD80staining Thus, both the 50 and 100 IU/kg doses of rFVIIIFc showed lowerimmunogenicity and antibody production in this model.

Introduction

Development of inhibitors to FVIII is recognized as a seriouscomplication in the management of hemophilia A. The incidence ofinhibitor formation is estimated to range from 20% to 30% in allhemophilia A to 30-40% in severe disease. (Green, Haemophilia 17:831-838(2011); Eckhardt et al. J. Thromb. Haemost. 9:1948-58 (2011)). Inhibitorpositive disease is currently managed by immune tolerance inductioninvolving the frequent high-dose administration of FVIII. Mechanisms ofinhibitor formation in patients are largely unknown and depend on amultitude of risk factors and cells and molecules of the immune system.

Inhibitor development in hemophilia involves a complex interplay ofmultiple cell types, surface molecules and secreted proteins of theimmune system including, T-lymphocytes, B-lymphocytes, antigenpresenting cells (APC; dendritic cells and macrophages), cytokines, andregulatory components of these cell types. Antibody production byB-cells depends on optimal help from T-cells, which are activated byantigen presentation from APC.

Tolerance to injected therapeutic peptides and proteins includingrecombinant FVIII is mediated by a class of T-cells called regulatoryT-cells (Treg) (Cao et al., J. Thromb. Haemost. 7(S1):88-91 (2009)).Several key molecules have been identified that correlate with inhibitorformation in hemophilia patients. These include the pro-inflammatorycytokine TNF-α, the anti-inflammatory cytokine interleukin (IL)-10, andthe Treg marker CTLA4, to name a few (Astermark et al., J. Thromb.Haemost. 5:263-5 (2007); Pavlova et al. J. Thromb. Haemost. 7:2006-15(2009)).

In this study, we investigated the splenic lymphocyte response torecombinant factor VIII Fc fusion protein (rFVIIIFc) compared withcommercially available full-length rFVIII (fl-rFVIII; ADVATE®) andB-domain deleted rFVIII (BDD-rFVIII; XYNTHA®/REFACTO AF®).

Materials and Methods

Materials:

Factor FVIII-deficient mice (Bi et al. Nat Genet. 10:119-21 (1995)) wereoriginally acquired from Dr. Kazazian (University of Pennsylvania,Philadelphia, Pa.) and maintained as breeding colonies either at BiogenIdec Hemophilia or Charles River Laboratory.

Antibodies used for staining and FACS analysis were obtained either fromBD Biosciences (Franklin Lakes, N.J., USA) or eBioscience (San Diego,Calif., USA). Antibodies used were directed against murine surfacemarkers such as CD4 (T-helper cells), CD11c and CD80 (dendritic cells),PD-1, PD-L1, CD25 (Treg cells), intracellular cytokines (IL-2, IL-4,TNF-α), and transcription factors (Foxp3).

Immunogenicity Study Design:

Three treatment groups received intravenous doses of 50, 100, or 250IU/kg, which were administered on days (FIG. 37). Each treatment anddose level were administered to 10 mice. Animals were euthanised on day56 by CO2 inhalation and spleens were dissected in sterile PBS.

Splenocytes were separated using the mouse spleen dissociation kit and agentle MACS dissociator (Miltenyi Biotec, Cologne, Germany). Single cellsuspensions of splenocytes were either fixed in 3% formalin for FACSstaining or stored in dissociation buffer for RNA isolation (Roche).

Assessments:

Anti-FVIII antibodies were determined using an in-house developed ELISA.Briefly, FVIII was coated on 96-well plates and used to captureantibodies from mouse plasma collected at specific time points.FVIII-specific antibodies were detected using a secondary anti-mouse IgGantibody. T-cell response profiling was conducted on isolated mousesplenocytes (FIG. 38). Lymphocytes and dendritic cells in spleen werestained for surface and intracellular targets.

For surface staining, 1×10⁶ total splenocytes were incubated withantibodies at appropriate concentrations. For intracellular staining,cells were permeabilised with BD Fix-Perm solution (BD Biosciences)followed by incubation with antibodies to cytokines in the same buffer.Foxp3 staining was carried out using antibody in Foxp3 staining buffer(BD Biosciences). Fluorescence intensity was recorded using a BD FACSCanto II and analysis performed using FLOWJO® software.

Lymphocytes were costained with CD4 and intracellular markers IL-2,TNF-α, and IL-4. Treg cells were stained for surface markers CD4 andCD25 followed by intracellular Foxp3. Splenocytes were stained for CD11cand PD-L1 (dendritic cells) or CD4 and PD-1 (CD4+ T-cells) to identifycells involved in the PD-L1-PD-1 pathway. Total RNA was isolated (Roche)and reverse transcribed to cDNA (Qiagen, Hilden, Germany). Primers forTGF-β, IL-10, IL-12a, and EBI-3 were purchased from IDT technologies.SYBR green-based real-time polymerase chain reaction (PCR) was carriedout using Quantitect system (Qiagen) using an ABI 7900 Fast Blockreal-time PCR machine. Data were analysed using the 7500 softwareversion 2.0.5.

Results

Total Anti-FVIII Antibody Levels on Day 42:

Total anti-FVIII IgG levels on day 42 were assayed from plasma ofhaemophilia A mice injected with 50, 100 or 250 IU/kg of rFVIIIFc,BDD-FVIII (XYNTHA®) or full length FVIII (fl-rFVIII) (ADVATE®) usingELISA. At both 50 and 100 IU/kg the rFVIIIFc group had significantlylower antibody levels compared to BDD-rFVIII and full length fl-FVIII,which indicated lower antigenicity to FVIII imparted by rFVIIIFcinjections. At 250 IU/kg the groups were not significantly differentfrom each other and had high antibody levels (FIG. 39).

Intracellular Cytokines (IL-2 and TNF-α) in CD4+ Cells:

Mice receiving 50 IU/kg in each treatment group were nonresponders basedon antibody levels to FVIII, whereas the mice receiving 250 IU/kg ineach treatment group were responders with the highest antibody levels(data not shown). rFVIIIFc at 50 and 100 IU/kg doses (FIG. 40A and FIG.40B) lowered the percentage of IL-2 and TNF-α positive CD4+ cells, whichindicated lower immunogenicity whereas BDD rFVIII and full-length rFVIIIshowed higher cytokine positive cells. All 3 treatments at the 250 IU/kgdose (FIG. 40C) elevated the percentage of cytokine positive cells,which indicated higher immunogenicity at this dose. Similar results wereobtained for IL-4.

CD4/CD25/Foxp3 Triple Positive Cells (Markers for Treg Cells):

At the 100 IU/kg dose, the percentage increase of Treg cells overvehicle were significantly higher in the rFVIIIFc group (P<0.05)compared with BDD-rFVIII and fl-rFVIII groups (FIG. 41). Similar resultswere obtained for the 50 IU/kg of rFVIIIFc indicating that both the 50and 100 IU/kg rFVIIIFc treatments can promote predominance of Treg cellsand suppress immune responses to FVIII.

Real-Time Polymerase Chain Reaction for Tolerance-Related Cytokines:

Real Time PCR analysis for immuno tolerance related cytokines namelyTGF-β (FIG. 42A), IL-10 (FIG. 42B) and IL-35 (IL-12a and EBI-3 subunits,shown respectively in FIGS. 42C and 42D) was carried out using RNAisolated from mice belonging to the 100 IU/kg treatment group. mRNAlevels were upregulated for the tested cytokines in the rFVIIIFc group(P<0.05) compared with the other treatments at 100 IU/kg, whichindicated the presence of splenocytes actively expressing tolerogeniccytokines thereby promoting an immunosuppressive microenvironment. mRNAexpression immunotolerance markers Foxp3 (FIG. 42E), CD25 (FIG. 42F),CTLA-4 (FIG. 42G), and Indoleamine 2,3-dioxygenase (IDO-1) (FIG. 42H)were higher in total splenocytes from 100 IU/kg group

FACS Analysis of PD-L1-PD-1 Pathway:

PD-L1 (CD274) on dendritic cells engage PD-1 (CD279) on T-cells topromote inhibitory pathways that suppress T-cell activation andproliferation, thereby leading to suppression of immune responses.Splenocytes from the 100 IU/kg group were stained for either surfaceCD11c and PD-L1 (FIG. 43A), or CD4 and PD-1 (FIG. 43B). At the 100 IU/kgdose, the percentage of dendritic cells positive for PD-L1 and thepercentage of T-cells positive for PD-1 were higher in the animalsreceiving rFVIIIFc (P<−0.05) compared with those receiving BDD-FVIII andfl-rFVIII. This indicated positive regulation of immunosuppressivepathways at both dendritic cells and T-cells by rFVIIIFc.

Discussion

The experimental results shown above indicated that rFVIIIFc at doses of50 and 100 IU/kg had low immunogenicity and promoted tolerance to FVIII,as demonstrated by:

(a) A lower level of pro-immunogenic cytokines (IL-2 and TNF-α) in CD4+T-cells compared with other FVIII molecules;

(b) An up-regulation of regulatory T-cells and markers (Foxp3, CD25,PD-1, CTLA4) that are responsible for immune tolerance inrFVIIIFc-injected mice. The significance of Foxp3+ Treg and the role ofCTLA4 in promoting tolerance to FVIII in haemophilia have beenpreviously described (Cao et al., J. Thromb. Haemost. 7(S1):88-91(2009); Astermark et al. J. Thromb. Haemost. 5:263-5 (2007)).(c) A higher level of tolerogenic cytokines 01-10, TGF-β, IL-35) insplenocytes of mice injected with rFVIII. These markers have been shownin several studies to be key immunoregulatory cytokines and majordeterminants of immuno-tolerance (Bi et al., Nat. Genet. 10:119-21(1995)).(d) An elevation of tolerogenic dendritic cell population (PD-L1, IDO-1,and decreased CD80) in mice following injection with rFVIIIFc.Concurrently, rFVIIIFc-treated mice also showed lower or no antibodiesto FVIII at doses of 50 and 100 IU/kg (Liu et al., WFH Abstract#FB-WE-04.2-5 (2012)) and resisted challenge with 250 IU/kg oncetolerised wuth 50 IU/kg of rFVIIIFc.

Conclusions:

rFVIIIFc-treated mice showed lower or no antibodies to FVIII at doses of50 and 100 IU/kg compared with traditional FVIII therapies. Based onT-cell and dendritic cell studies in mice, rFVIIIFc was found to be lessimmunogenic than traditional FVIII therapies and promoted tolerogenicpathways. rFVIIIFc upregulated key immunoregulatory cytokines insplenocytes of haemophilia A mice that are indicative ofimmunotolerance. Taken together these findings indicate the existence ofa tolerogenic microenvironment in the spleen of mice injected withrFVIIIFc at low doses (50 and 100 IU/kg).

These studies have demonstrated for the first time that rFVIIIFcactivates dendritic cell signaling, which is a crucial determinant ofimmunotolerance. These findings indicate the existence of functionalimmune tolerance to FVIII imparted by rFVIIIFc in haemophilia A mice.

Example 15 Evaluation of Antibody Responses to rFVIIIFc Compared toXYNTHA® and ADVATE® in Hemophilia A Mice

Development of inhibitory antibodies to replacement FVIII occurs in20-30% of previously untreated patients, being the most severecomplication of hemophilia treatment. Immune tolerance induction (ITI),which entails frequent administration of FVIII, is currently used totreat patients who develop inhibitors. A subset of these patients,however, do not respond to ITI. See, e.g., Green, Haemophilia 17:831-838(2011); Eckhardt et al. J. Thromb. Haemost. 9:1948-58 (2011); Cao etal., J. Thromb. Haemost. 7(S1):88-91 (2009). Recombinant FVIIIFc has ahalf-life approximately 1.6-fold longer than the half-life of rFVIII andit is currently in phase 2/3 clinical development. The experimentsdisclosed herein assess the immunogenicity and immune toleranceproperties of rFVIIIFc compared with other rFVIII replacement proteinsin hemophilia A (HemA) mice.

(a) Immunogenicity Comparison for rFVIIIFc, XYNTHA® and ADVATE® in HemAMice: Antibody Response:

Four groups of HemA mice, with 9-12 HemA mice per group, were treatedwith 50 IU/kg, 100 IU/kg and 250 IU/kg doses of rFVIIIFc, XYNTHA®,ADVATE®, and vehicle control. Doses were administered at day 0, day 7,day 14, day 21, and day 35. Blood was drawn at day 0, day 14, day 21,day 28 and day 42 (FIG. 44).

rFVIIIFc induced a significantly lower antibody response at 50 IU/kg(FIG. 45) and at 100 IU/kg (FIG. 46) compared to XYNTHA® and ADVATE®.However, all FVIII proteins showed similar antibody response at 250IU/kg (FIG. 47). Neutralizing antibody titers correlated with totalbinding antibody levels (FIG. 48).

(b) Immunogenicity Comparison for rFVIIIFc, XYNTHA® and ADVATE® in HemAMice: T-Cell Response Profiling:

For T-cell response profiling in splenocytes, an additional dose ofrFVIIIFc, XYNTHA®, ADVATE®, and vehicle control was administered at day53. Spleens were collected at day 56 (FIG. 49). The results indicatedthat rFVIIIFc promotes predominance of CD4/CD25/Foxp3-positive TregCells (FIG. 50, right panel).

Summary:

Administration of rFVIIIFc resulted in significantly lower antibodyresponse compared with XYNTHA® and ADVATE® at doses of 50 and 100 IU/kg.The tolerogenic profile following 50 and 100 IU/kg doses of rFVIIIFcindicated that rFVIIIFc can promote predominance of Treg cells andsuppress immune response to FVIII

(c) rFVIII Immune Tolerization Study:

To test whether repeated administration of rFVIIIFc can inducefunctional tolerance in vivo the following dosing regimen was adopted.HemA mice (8-10 wk old) were injected with 50 IU/kg of rFVIIIFc orvehicle every week for 4 weeks (on days 0, 7, 14, 21) followed by oneinjection on day 35. Starting day 49 these mice were challenged withweekly 250 IU/kg of rFVIIIFc to determine if the animals can toleratehigh doses of rFVIIIFc. The challenge doses were administered on days 0,7, 14, and 21 starting day 49 of the study (see FIG. 51). Blood sampleswere collected at specified time points to check for anti-FVIIIantibodies using ELISA. As shown in FIG. 52, repeated dosing of rFVIIIFcled to statistically significant reduction in antibodies to rFVIIIFcwhen challenged at high doses of 250 IU/kg, while animals receivingvehicle had higher levels of antibodies upon challenge. This clearlyindicates that repeated administration of rFVIIIFc at 50 IU/kg based onthe dosing scheme followed can induce tolerance to higher doses ofrFVIIIFc (250 IU/kg).

Conclusion:

At therapeutic doses, rFVIIIFc was found to be (1) less immunogeniccompared with XYNTHA® and ADVATE®, and (2) capable of inducing immunetolerance to FVIII in HemA mice.

Currently, its being determined whether even lower doses of rFVIIIFc canlead to immune tolerization to higher doses of rFVIIIFc. In the presentstudy, lower doses of rFVIIIFc, namely 25 IU/kg and 10 IU/kg, are beingused during the tolerance induction phase. This will be followed bychallenging with 250 IU/kg of rFVIIIFc and measurement of antibodies toFVIII as carried out for the previous study.

Example 16 Clearance Pathways of rFVIIIFc in Haemophilia A Mice

Long-lasting recombinant coagulation FVIII Fc fusion protein (rFVIIIFc)is currently in phase 3 clinical study for episodic and prophylactictreatment of individuals with haemophilia A. Compared with recombinantfull-length FVIII (ADVATE®, Baxter Healthcare Corporation), rFVIIIFc hasa 1.7-fold extended half-life and significantly decreased clearance inpatients with haemophilia A. See, Powell et al., Blood 119:3031-7(2012). This improved pharmacokinetic (PK) profile is mediated throughthe interaction of Fc with neonatal Fc receptor (FcRn). See Dumont etal., Blood 119:3024-30 (2012). rFVIIIFc is comprised of a singleB-domain deleted human coagulation FVIII attached directly to the Fcdomain of human immunoglobulin G1, which is naturally recycled uponcellular uptake (endocytosis or pinocytosis) through interaction withFcRn (FIG. 53). Monocytic cells (macrophages and dendritic cells),including liver resident macrophages (Kupffer cells), are implicated inthe clearance of von Willebrand factor (VWF) and FVIII (FIG. 54). Seevan Schooten et al., Blood 112(5):1704-12 (2008).

In order to elucidate the cell types involved in rFVIIIFc uptake,clearance, and FcRn-mediated recycling, we studied the impact ofmacrophage and Kupffer cell depletion on the clearance of rFVIIIFc ingenetically engineered mouse models.

Materials and Methods

The clearance of rFVIIIFc and rFVIII (BDD) was compared in 3 knockout(KO) mouse models: (1) haemophilia A (FVIII KO), deficient in FVIII; (2)double KO (DKO) of FVIII and VWF, lacking expression of both FVIII andVWF; and (3) FcRn-KO, lacking expression of the FcRn. In all 3 models,macrophage and Kupffer cells were depleted with CLODROSOME® (EncapsulaNanoSciences, Inc), which is a toxic ATP analogue (clodronate)encapsulated in liposomes that is specifically phagocytosed bymacrophages and triggers apoptosis. See van Rooijen & Hendrikx, MethodsMol. Biol. 605:189-203 (2010).

Control mice in each group were treated with ENCAPOSOME® nontoxicliposomes (FIG. 55). A single intravenous dose of either FVIII orrFVIIIFc (125 or 250 IU/kg) was injected 24 hours after treatment withliposomes. Blood samples were collected by retroorbital or vena cavablood collection at specified time points (4 samples per time point).

Human FVIII activity in plasma samples was then measured by a FVIIIchromogenic assay, and PK parameters were estimated with WINNONLIN®software (Pharsight Corp.) using a noncompartmental analysis model.

Kupffer cell and macrophage depletion was evaluated byimmunohistochemical staining, and was quantified using Visiopharmsoftware (Hoersholm, Denmark) or by reverse transcrition polymerasechain reaction (RT-PCR).

Results

(a) Depletions of Macrophages and Kupffer Cells:

FIG. 56 shows a representative staining of liver sections with anantibody to Iba-1, a specific macrophage marker, 24 hours after controlENCAPSOME® (A, A′) or CLODROSOME® (B. B′) treatment of haemophilia Amice. A′ and B′ show the quantification masks highlighting the stainedKupffer cells (azure), total tissue area (navy blue), and empty areas(grey). Similar depletion profiles were obtained in other mouse strains.Quantitative analysis of positively stained areas showed that >90% ofliver Kupffer cells were depleted and remained low for >3 days postCLODROSOME® treatment (n=4) compared with control ENCAPSOME®-treatedanimals (FIG. 57). Circulating monocytic cells were also reduced by >50%within 24 hours of CLODROSOME® treatment, assessed by flow cytometricanalysis of blood cells stained with a labeled antibody to F4/80+ (n=4).Within 48 hours, depleted blood cells recovered (FIG. 57). Consistentwith the observed depletion of macrophages in the liver, RT-PCR analysisof the expression of the macrophage marker epidermal growth factormodule-containing mucin-like receptor 1 (Emr1) (F4/80) showed thatCLODROSOME® treatment decreased Emr1 mRNA expression >95% in liver andlung in haemophilia A mice (FIG. 58). Emr1 is the designation used forthe human protein. The mouse homolog is known as F4/80. Emr1 is atransmembrane protein present on the cell-surface of mature macrophages.

(b) Clearance of FVIII in Mouse Models:

In contrast to previously reported results that suggest Kupffer cellsplay a role in the clearance of both FVIII and VWF (van Schooten C J, etal. Blood. 112(5):1704-12 (2008)), Kupffer cell depletion did not causethe expected decrease in FVIII clearance in haemophilia A mice (FIG.59). To the contrary, depletion of Kupffer cells in haemophilia A micesignificantly increased the clearance of rFVIIIFc (FIG. 59). Similarly,in DKO mice, Kupffer cell depletion did not decrease FVIII clearance andsignificantly increased clearance of rFVIIIFc (FIG. 60). In FcRn-KOmice, Kupffer cell depletion did not affect the clearance of FVIII orrFVIIIFc (FIG. 61).

Conclusions:

Macrophage and Kupffer cell depletion in haemophilia A and DKO mice(deficient in FVIII and VWF) increased rFVIIIFc clearance, but did notdecrease FVIII clearance, indicating that macrophage and Kupffer cellscan account for much of the difference in clearance between FVIII andrFVIIIFc in these mice. In the absence of both VWF and FVIII (DKO mice),depletion of macrophages and Kupffer cells increased the clearance ofrFVIIIFc to levels approaching that of FVIII.

The lack of effect of Kupffer cells depletion on FVIII clearance ineither haemophilia A or DKO mice contrasted with previously publishedstudies. See van Rooijen & Hendrikx, Methods Mol. Biol. 605:189-203(2010). Macrophage and Kupffer cell depletion in FcRn-KO mice did notresult in significant clearance differences between FVIII and rFVIIIFc,indicating a potential role of FcRn in macrophage-mediated recycling ofrFVIIIFc.

Together, these studies indicated that rFVIIIFc was protected in amacrophage- and/or Kupffer cell-dependent manner and that a naturalpathway in these cells can contribute to FcRn-mediated recycling ofrFVIIIFc.

Example 17 Immune Tolerization to Pre-Existing Antibodies to Factor VIII

It is highly likely that patients that receive rFVIIIFc were previouslyon a different rFVIII therapy. About 30% of hemophilia A patientsdevelop antibodies to FVIII, which is a major problem in current FVIIIreplacement therapy. Currently, the only approved approach to tolerizepatients having FVIII inhibitors is to dose them with high doses ofFVIII for an indefinite period of time, i.e., until the patient istolerized. Thus, it is of interest to determine if low dose rFVIIIFc canreverse the immunogenicity of FVIII by inhibiting T and B cell signalingpathways and skewing towards tolerance.

In this study, hemA mice (8-10 wks old) will be injected weekly once for5 weeks with either BDD-FVIII (XYNTHA®) or fl-FVIII (ADVATE®) at 50IU/kg. Levels of anti-FVIII antibodies will be determined in bloodsamples drawn from these animals on specified days as indicated in thescheme. Following the induction of inhibitory antibodies, animals willbe switched to injections with 50 IU/kg of rFVIIIFc weekly for 5 weeks.This will be the tolerization phase. Levels of anti-FVIII antibodieswill be determined again from blood samples drawn as indicated.Following the tolerization phase, animals will be injected again withXYNTHA® or ADVATE® at 50 IU/kg for another four injections and bloodsamples drawn will be tested for anti-FVIII antibodies. In the event ofsuccessful tolerization, the second challenge with XYNTHA® or ADVATE®should not produce any antibodies to FVIII which may indicate thecapability of rFVIIIFc to induce immunotolerance to FVIII.

Example 18 T-Cell Adoptive Immunotherapy and Transfer of ImmuneTolerance

Regulatory T-cells (Tregs) are the master players in the induction andmaintenance of peripheral tolerance to FVIII and other peptidetherapeutics. One of the key studies used in determining the presence offunctional immune tolerance is by transferring Treg cells isolated fromtolerized animals to recipients, which are then challenged with higherdoses of FVIII. The presence of functional transfer of tolerance isevidenced by lack of or lesser antibody production in the challengedrecipient mice.

In this study, hemA mice (8-10 weeks old) will be tolerized by injecting50 IU/kg of rFVIIIFc or vehicle every week for 4 weeks (on days 0, 7,14, 21) followed by two injections on days 35 and 53. Plasma sampleswill be collected for determination of anti-FVIII antibody levels usingELISA. On day 56, mice with no antibodies will be euthanized and theirspleens will be isolated. Splenocytes from these mice will be harvestedand made into single cell suspensions using the splenocyte isolation kit(Miltenyi Biotec). Tregs from total splenocytes will be isolated usingthe CD4 CD25 murine Treg magnetic bead based isolation kit (MiltenyiBiotec). Isolated Tregs will be enumerated using a cell counter. Analiquot of the cell suspension will be fixed using 3% formalin forphenotypic analysis by FACS to determine the purity of the isolate.Tregs will then be injected into recipient mice on day 0 at 1×10⁶cells/mouse in 200 μL saline by IV.

Starting day 1 animals will be challenged with 250 IU/kg of rFVIIIFcfollowed by repeated injections weekly once on days 8, 15, 22, and 29.Blood samples will be collected on days 15, 22, 29 and 36 for plasmaanti-FVIII antibody analysis using ELISA. In the event of successfultransfer of tolerance, mice receiving Tregs from rFVIIIFc tolerizedanimals will not develop antibodies to FVIII whereas, mice injected withTregs isolated from vehicle injected donor mice show the presence ofanti-FVIII antibodies.

Example 19 Identification of Mechanisms of Immune Tolerance Induction byrFVIIIFc: Studies with Dendritic Cells and Macrophages

One of the key features that distinguishes rFVIIIFc from other FVIIIdrug products is the presence of the Fc moiety which is a naturallyoccurring constituent. This could be one responsible factor thatsuppresses the immune response and drives an immune tolerance reaction.Fc molecules present in IgG and in rFVIIIFc are capable of interactingwith the classical IgG Fc receptors and FcRn. Among the Fc receptors,the subtype FcγRIIb (FcγR2b) is an inhibitory receptor and deliverssuppressive signals that curb the activation of cell types harboringthat receptor. Fc receptors including FcRn are chiefly localized inantigen presenting cells (APC—dendritic cells, macrophages and B-cells),but not in T-cells. Therefore, it is possible that the rFVIIIFc couldengage FcR2b and/or FcRn in these cells to skew towards a tolerogenicphenotype.

To ascertain whether rFVIIIFc engages FcR2b and/or FcRn in dendriticcells and macrophages to develop a tolerogenic phenotype, the followingexperiments will be conducted:

1. Identification of markers at the mRNA and cell surface levels(protein) in murine macrophage cell lines, splenic macrophages andsplenic dendritic cells that are regulated by rFVIIIFc in comparisonwith FVIII alone or a mutant of FVIIIFc (N297A) which does not interactwith Fc receptors.

2. Overexpression and knockdown of FcgR2b or FcRn in RAW 264.7 murinemacrophage cell line and investigating the effects of rFVIIIFc on thesereceptors by studying downstream targets

3. Identification of the possible involvement of other pathways ofimportance such as TLR mediated signaling in the APC response torFVIIIFc

Example 20 Alternate Routes for Induction of Tolerance

Immune tolerance to rFVIIIFc may also be achieved via mucosal route. Twopossible sites for inducing tolerance are the gastrointestinal mucosaand the respiratory mucosa. Oral tolerance to rFVIIIFc can be induced byeither feeding animals or using oral gavages to deliver the molecule tothe intestinal mucosa. The gut mucosa has specialized secondary lymphoidorgans namely the Peyer's patches which contains antigen presentingcells (APC) that are important in regulatory immune responses. TheseAPCs can process antigens and activate specific subsets of dendriticcells and Tregs which can travel to other sites in the body and programother cells of the immune system to suppress an immune response in thepresence of antigen, in this case exogenously supplied FVIII. Thisphenomenon could be mediated by Fc interactions with FcRn and/or FcgR2bpresent on the APC of the gut mucosal immune system. Respiratory mucosacan be tolerized by inhalation of aerosols of rFVIIIFc which may operatebased on the same mechanism as for the gut.

The ascertain whether immune tolerance can be achieve via mucosal route,XYNTHA® or ADVATE® will be administered orally or via aerosol to hemAmice. Following the induction of inhibitory antibodies, animals will beswitched to rFVIIIFc. This will be the tolerization phase. Levels ofanti-FVIII antibodies will be determined from blood samples drawn asindicated. Following the tolerization phase, animals will be injectedagain with XYNTHA® or ADVATE® and blood samples drawn will be tested foranti-FVIII antibodies. In the event of successful tolerization, thesecond challenge with XYNTHA® or ADVATE® should not produce anyantibodies to FVIII which may indicate the capability of rFVIIIFc toinduce immunotolerance to FVIII after mucosal administration.

Example 21 Immunotolerance to Other Clotting Factors

To ascertain whether immunotolerance can be conferred by Fc to clottingfactor payloads other than FVIII, chimeric polypeptides comprising theFVIIa clotting factor fused to Fc (rFVIIaFc), or the FIX clotting factorfused to Fc (rFIXFc) are generated. Cloning, expression, andpurification of rFVIIaFc and rFIXFc are performed according to methodsknown in the art. Biochemical, biophysical and pharmacokineticcharacterization of rFVIIaFc and rFIXFc are conducted as disclosed inthe examples above and/or using methods known in the art. The evaluationof antibody responses to rFVIIaFc and rFIXFc compared to commercialclotting factors and immunotolerance studies are performed as disclosedin the examples above. Chimeric polypeptides comprising a clottingfactor payload and an Fc moiety such as rFIXFc or FVIIaFc caneffectively induce immunotolerance to the unmodified clotting factor(i.e., FIX or FVII without an Fc portion).

Example 22 Transplacental Transfer of Immune Tolerance to FVIII UsingrFVIIIFc

The objective of this study was to determine whether the administrationof rFVIIIFc to pregnant mice could transfer the molecule to the fetusvia the placenta (due to the interaction of the Fc portion with the FcRnreceptor on placental cells), and whether the exposure of the fetalimmune system to the rFVIIIFc at an early stage could lead to toleranceby programming the thymus to recognize FVIII as a self antigen and notto develop an immune reaction towards it.

Pregnant mice were infused with either one dose (6 U) of rFVIIIFc orXYNTHA® intravenously by retro-orbital injection on day 16 of gestationor two doses (6 U each) by tail vein injection on days 15 and 17 ofgestation. The immunogenicity of XYNTHA® was evaluated in the pups bornout of the immunized mothers, after pups had reached maturity (ages 6-9weeks old). Day 16 of pregnancy was chosen because it coincided with thedevelopment of the autoimmune regulatory molecule, AIRE, which plays acrucial role in removing self-reactive T-cells from the thymus. Theresults showed that in the first experiment, that tested dosing ofpregnant mice with a single infusion of rFVIIIFc or XYNTHA® at day 16 ofgestation, pups born out of rFVIIIFc treated pregnant mice hadstatistically significantly lower Bethesda titers to XYNTHA®, comparedto pups born out of XYNTHA® treated pregnant mice (FIG. 62). In thesecond experiment, that tested dosing of pregnant mice on d 15 and d17of gestation, pups born out of rFVIIIFc treated pregnant mice hadstatistically significantly lower Bethesda titers to XYNTHA®, comparedto pups born out of control mothers and a trend for lower titerscompared to pups born from mothers treated with XYNTHA® (FIG. 63)

The transplacental transfer of protein (rFVIIIFc) from maternal to thefetal circulation will be evaluated by detecting FVIII activity in pupsborn out of immunized mice. The actions at the T-cell level will beevaluated by T-cell profiling and Treg transfer studies from rFVIIIFctolerized pups.

Example 23 rFVIIIFc Regulates Tolerogenic Markers in Antigen PresentingCells

Materials and Methods:

Mice:

Hemophilia A (HemA) mice (C57BL/6) bearing a FVIII exon 16 knockout on a129×B6 background (Bi, L., Lawler, A. M., Antonarakis, S. E., High, K.A., Gearhart, J. D., and Kazazian, H. H., Jr. 1995. Targeted disruptionof the mouse factor VIII gene produces a model of haemophilia A. NatGenet 10:119-121) were obtained from Dr. H. Kazazian (University ofPennsylvania, Philadelphia, Pa., USA) and maintained as breedingcolonies at either Biogen Idec Hemophilia or Charles River Laboratory orJackson Laboratories. All animal procedures used were approved by theInstitutional Animal Care and Use Committee (IACUC) and performed basedon guidelines from the Guide to the Care and Use of Laboratory Animals.

Antibodies and Reagents:

Antibodies used for staining and Flow Cytometry were obtained from BDBiosciences (Franklin Lakes, N.J., USA) or eBioscience (San Diego,Calif., USA). Antibodies used were directed against murine surfacemarkers such as CD4 and PD-1 (T-helper cells), CD11c, CD80, and PD-L1(dendritic cells), and CD25 (Treg cells), intracellular cytokines (IL-2,IL-4, TNF-α), and transcription factors (Foxp3). Reagents forintracellular staining for cytokines and transcription factors werepurchased from BD Biosciences. Recombinant B-domain deleted FVIIIFc(rFVIIIFc) and recombinant B-domain deleted FVIII (rFVIII) weresynthesized as in Peters et al. (Peters, R. T., Toby, G., Lu, Q., Liu,T., Kulman, J. D., Low, S. C., Bitonti, A. J., and Pierce, G. F. 2012.Biochemical and functional characterization of a recombinant monomericFactor VIII-Fc fusion protein. J. Thromb Haemost. DOI:10.1111/jth.12076).

Other Factor VIII drug substances namely rBDD FVIII XYNTHA® (WyethPharmaceuticals, Philadelphia, Pa., USA), and full-length FVIII ADVATE®(Baxter Healthcare Corporation, Westlake Village, Calif., USA) werepurchased and reconstituted according to manufacturers' guidelines.

Immunization/Tolerance Induction in Mice:

Three treatment groups consisting of 8-10 week old male HemA micereceived intravenous doses of 50, 100, or 250 IU/kg, which wereadministered on days 0, 7, 14, 21, 35, and 53. Each treatment and doselevel was administered to 10 mice. Blood samples were collected byretro-orbital bleeding on days 0 (pre-bleed), 14, 21, 28, and 42, andused for separating plasma and determining anti-FVIII antibody levelsusing ELISA. Animals were injected once more on day 53, euthanised onday 56 by CO₂ inhalation and spleens were dissected in sterile PBS.Splenocytes were separated using the mouse spleen dissociation kit and agentle MACS dissociator (Miltenyi Biotec, Cologne, Germany). Single cellsuspensions of splenocytes were either fixed in 3% formalin (BostonBioProducts) for FACS staining or stored in dissociation buffer for RNAisolation (Roche Applied Science, Indianapolis, Ind.). For immunetolerance testing studies, mice were initially injected with 50 IU/kg ondays 0, 7, 14, 21, and 35. After confirming the absence of antibodies toFVIII on day 42, these mice were administered with 250 IU/kg of rFVIIIFconce weekly; i.e., days 49, 56, 63, and 70 (days 0, 7, 14, and 21 ofrechallenge). Rechallenged animals were tested for anti-FVIII antibodylevels on bleeds collected on days 14, 21, and 28.

Anti-FVIII Antibody ELISA:

The protocol followed was designed in house at Biogen Idec Hemophilia.Prior to the assay, all plasma samples were warmed to 56° C. in a waterbath to inactivate residual coagulation factors introduced by thetreatments and anti-coagulation enzymes that could break down the coatedstandards on the plate. On day 1, a 96 well, high binding microtiterplate (Thermo Immulon 2HB) was coated with 1 μg/ml (100 μl/well)B-domain deleted FVIII in 0.05M sodium carbonate, pH 9.6 and incubatedovernight (12 to 18 hours) at 4° C. On the following day the supernatantwas removed and the plate washed 4 times with PBST (phosphate bufferedsaline containing 0.05% Tween 20). The plate was then blocked with 200μl per well of PBST containing 10% heat inactivated horse serum, pH 7.4for 60 minutes at room temperature. The standard used for mouse IgG wasa polyclonal pool of anti-FVIII monoclonal antibodies prepared by mixingequal amount of GMA8002 (A1), GMA8008 (C2), GMA8011(C1), GMA8015(A2),GMA8016 (A2), GMA8005 (A1/A3). All monoclonal antibodies were from GreenMountain Antibodies, Inc, Burlington, Vt. with the FVIII domain epitopesare shown in parenthesis. Mouse plasma test samples were diluted inblocking buffer and contained the same concentration of heated plasma asthe standards. The blocking buffer was then removed and the dilutedstandards and samples were added at 100 μl/well in duplicates. The platewas incubated for 2 hours at 37° C. with shaking on an orbital shaker.After washing 4 times with PBST 100 μl/well of goat anti-mouse IgG-HRP,diluted 1:20000 in blocking buffer was added and incubated for 60minutes at 37° C. with shaking on an orbital shaker. The plate waswashed again 4 times with PBST, and then 100 μl/well of TMB was addedand incubated at RT for 5 to 10 minutes. Results were read at OD 650with a plate reader

Bethesda Assay for Determining Neutralizing Antibody Titers:

This assay determined the titer of neutralizing anti-FVIII antibodies ina given plasma sample. Briefly, the assay was performed by mixing plasmasamples with known concentrations of recombinant FVIII and incubation at2 hours at 37° C. Residual FVIII activity in the mixture was then testedusing a Coatest FVIII SP kit, in the presence of Factor IXa, Factor X,phospholipids and CaCl2. The activity of FVIII was calculated using astandard curve for FVIII activity plotted using rFVIII in the absence ofany inhibitors.

FACS Staining:

T-cell and dendritic cell response profiling was conducted on isolatedmouse splenocytes. Splenic lymphocytes and dendritic cells were stainedfor surface and intracellular targets. For surface staining, 1×10⁶ totalsplenocytes were incubated with antibodies at appropriateconcentrations. For intracellular staining, cells were permeabilisedwith BD Fix-Perm solution (BD Biosciences) followed by incubation withantibodies to cytokines in the same buffer. Foxp3 staining was carriedout using antibody in Foxp3 staining buffer (BD Biosciences).Fluorescence intensity was recorded using a BD FACS Canto II andanalysis performed using FLOWJO software. Lymphocytes were costainedwith CD4 and intracellular markers IL-2, TNF-α, and IL-4. Regulatory Tcells (Treg) were stained for surface markers CD4 and CD25 followed byintracellular Foxp3. Splenocytes were stained for CD11c and PD-L1(dendritic cells) or CD4 and PD-1 (CD4+ T-cells) to identify cellsinvolved in the PD-L1-PD-1 pathway.

Real Time PCR and Real Time PCR Based Array Analysis:

Total RNA was isolated using an RNA isolation kit (Roche AppliedScience, Indianapolis, Ind.) and reverse transcribed to cDNA (Qiagen,Hilden, Germany). Real time PCR primers for the tested genes weredesigned using the online algorithm available on the IDT technologieswebsite (www.idtdna.com) and were purchased from IDT technologies(Coralville, Iowa). SYBR green-based real-time PCR was carried out usingQuantitect system (Qiagen, Hilden, Germany) in an ABI 7900 Fast Blockreal-time PCR machine (Applied Biosystems, Foster City, Calif.). Forreal time PCR based arrays, 500 ng of total cDNA was mixed with SYBRgreen based qPCR master mix, and aliquoted onto 96-well plate array(PAMM047Z, T-cell Anergy and Immune Tolerance PCR Array; SA Biosciences,Frederick, Md., USA). Reactions were carried out with an ABI 7900 FastBlock real-time PCR machine (Applied Biosystems, Foster City, Calif.).Results were analysed using the 7500 software version 2.0.5 and therelative levels of gene transcripts were measured with endogenoushousekeeping genes as control using the 2−ΔCt relative quantificationmethod (Livak, K. J., and Schmittgen, T. D. 2001. Analysis of relativegene expression data using real-time quantitative PCR and the 2(−DeltaDelta C(T)) Method. Methods 25:402-408.). The housekeeping genes usedfor normalization were GAPDH, HPRT, Hsp90ab, beta-actin, and GusB. Foreach sample average Ct values for the housekeeping genes were takentogether and used to calculate the ΔCt. mRNAs that displayed thresholdcycles (Ct) >35 were excluded from the analysis.

T-Cell Proliferation and Determination of Interferon-γ (IFN-γ) Levels:

HemA mice (8-10 weeks old) were injected with FVIII drug substances oncea week for 2 weeks. Seventy two hours post the second injection micewere euthanized and peritoneal macrophages were collected by lavageusing sterile PBS. Splenic T-cells were isolated using magnetic beadbased murine CD4+ T-cell isolation kit (Miltenyi Biotec, Germany).T-cells were labeled with 10 μM CFSE (Invitrogen, Carlsbad, Calif.) for15 minutes in warm PBS and plated in 96 well ultra-low adhesion plates(Corning) along with peritoneal macrophages at a density of 1×10⁶ cellsper ml for each. Cells were then incubated with 0.1, 1 and 10 nM ofrFVIII or vehicle or CD3/CD28 microbeads (positive control; MiltenyiBiotec) in X-VIVO 15 medium (Lonza) containing co-stimulatory antibodiesnamely anti-CD28 and anti-CD49d (BD Biosciences), for 96 hours at 37° C.At the end of the incubation, IFNγ levels in the culture supernatantwere measured using an ELISA kit from Meso Scale Devices (MSD). T-cellproliferation was determined by measuring CFSE fluorescence intensity(MFI) in T-cells gated based on forward and side scatters, using FACS(BD FACS CANTO II).

Statistical Analysis:

Statistical analysis of results were carried out either using unpairedstudent's T-test or Mann-Whitney's T-test. P-values <0.05 wereconsidered to be significant.

Results:

Dendritic cells are professional antigen presenting cells and harbor keymolecules and enzymes that are pivotal in skewing an immune responsetowards tolerance. Splenocytes from hemA mice injected with FVIII drugsubstances or vehicle were stained for CD11c and MHC Class II moleculesto gate the dendritic cells. Splenic composition of dendritic cellsexpressing markers such as CD80, a surface marker upregulated in maturedendritic cells indicating more antigen presentation and CD274 (PD-L1),the ligand for the PD-1 receptor, were identified by co-staining withspecific antibodies to these molecules. As shown in FIG. 63A,splenocytes from 100 IU/kg rFVIIIFc or BDD-FVIII injected HemA miceshowed a significant decrease in the percentage of dendritic cellsexpressing CD80, suggesting an abundance of immature dendritic cells.

Although mice receiving fl-FVIII showed a decrease in the percentage ofCD80+ dendritic cells compared to vehicle, it did not attain statisticalsignificance (FIG. 64A). The trend was similar for mice receiving the 50IU/kg dose of rFVIIIFc. At 250 IU/kg, all the three treatments showed noalterations in the percentage of CD80+ dendritic cells among thesplenocytes.

Results from FACS analysis for splenic dendritic cells expressing CD274(PD-L1), revealed that rFVIIIFc at 100 IU/kg enhanced the percentage ofthese cells compared to vehicle and other FVIII treatments (FIG. 64B).This molecule was also regulated at the mRNA level by rFVIIIFc asillustrated by real time PCR analysis (FIG. 64C). This suggests therFVIIIFc regulates the PD-L1:PD-1 pathway, one of the keyimmunosuppressive pathways, thereby reducing the immunogenicity toFVIII. In addition to CD274, rFVIIIFc also upregulated mRNA levels ofindoleamine 2,3-dioxygenase (IDO), a key enzyme that regulates T-cellproliferation and activation by affecting tryptophan metabolism (FIG.64D).

Example 24 rFVIIIFc at Low Doses Enhances the Expression of Markers ofImmune Tolerance and Anergy in Splenocytes

Experimental procedures were conducted according to the Materials andMethods described in Example 23.

In order to identify markers of immune tolerance induced by rFVIIIFc,real time PCR based arrays focused on genes specific for immunetolerance and anergy (SA Biosciences) was employed. mRNA isolated fromsplenocytes obtained from mice injected with 50 and 250 IU/kg ofrFVIIIFc and vehicle was used to identify novel response elementsactivated by this drug substance.

Candidates were identified based on their expression level (2-fold aboveor below vehicle), and the p-value based on student's T-test (<0.05).Relative expression of each gene was determined using the 2^(−ΔCt)method (Livak, K. J., and Schmittgen, T. D. 2001. Analysis of relativegene expression data using real-time quantitative PCR and the 2(−DeltaDelta C(T)) Method. Methods 25:402-408) after normalizing to the averageCt values of a set of 5 housekeeping genes (see Example 23, Materialsand Methods).

Based on these criteria, array analysis performed revealed candidatesthat were preferentially regulated by rFVIIIFc at 50 IU/kg at thesplenocyte level, in comparison to vehicle and 250 IU/kg injected mice(FIG. 65 and FIG. 66). These included statistically significantupregulation of tolerance specific genes such as Foxp3, CTLA-4, andCD25; anergy associated genes such as Egr2, Dgka, and CBL-B; genesbelonging to the Tumor Necrosis Factor Superfamily (TNFRSF);prostaglandin synthase 2 (PTGS2) and prostaglandin E2 receptor (PTGER2)(see FIG. 66). Conversely some of the downregulated genes identifiedusing this array are known pro-inflammatory molecules such as CCL3 andSTAT3 (FIG. 66).

These results indicated the presence of tolerogenic pathways and atolerogenic microenvironment within the splenocytes of animals receiving50 IU/kg of rFVIIIFc. The gene expression profiles determined using thearray will be validated using real time PCR with individual primer pairsfor the identified candidates.

Example 25 CD4+ Splenocytes from 250 IU/Kg Group of Mice Proliferate andProduce High Levels of IFN-γ

Experimental procedures were conducted according to the Materials andMethods described in Example 23.

T-cell proliferation studies were employed to investigate factor VIIIspecific T-cell responses ex vivo. CD4+ T-cells isolated from micereceiving two weekly injections of either 50 or 250 IU/kg of rFVIIIFc,were labeled with CFSE and reconstituted with peritoneal macrophages(antigen presenting cells; APC) and incubated in the presence ofB-domain deleted rFVIII at three concentrations, 0.1, 1, and 10 nM.

FACS analysis for proliferation based on CFSE dilution signals revealedthat, T-cells from the group treated with 250 IU/kg of rFVIIIFc showed adose dependent increase in proliferation, which was statisticallysignificant at 10 nM (FIG. 67). In parallel, T-cells from mice treatedwith 50 IU/kg of rFVIIIFc did not show a significant increase inproliferation with escalating concentrations of rFVIII ex vivo comparedto vehicle (FIG. 67).

IFN levels measured from the culture supernatants of these incubationsrevealed a similar profile matching the T-cell proliferation pattern,i.e., a dose dependent increase in secretion from T-cells derived frommice treated with 250 IU/kg of rFVIIIFc (FIG. 68A) whereas no changes inlevels from T-cells from mice treated with 50 IU/kg of rFVIIIFc (FIG.68B).

In order to dissect the mechanism(s) of action of rFVIIIFc insuppressing the immune response to FVIII, we employed two mutantconstructs—one which does not bind to the Fcγ receptor (termedrFVIIIFc-N297A) and the other one which lacks binding the to the FcRnreceptor (termed rFVIIIFc-IHH). These constructs were used to identifythe interactions of the rFVIIIFc with one of these receptors in bringingabout the immunosuppressive action. To this end, we tested the IFNγsecretion profile of T-cells derived from mice that received two weeklyinjections of rFVIIIFc-N297A at 250 IU/kg doses (FIG. 68C) and 50 IU/kgdoses (FIG. 68D).

Side by side comparisons of IFNγ secretion from T-cells derived from 250IU/kg of rFVIIIFc and rFVIIIFc-N297A revealed that the level of thecytokine was highly reduced in the T-cells from animals receiving themutant, albeit significantly higher levels compared to vehicle. However,the levels of IFNγ from the 50 IU/kg group of the mutant protein did notshow a significant difference from that of 50 IU/kg of rFVIIIFc. Thissuggests that the higher antibody production and T-cell proliferationobserved at higher doses of rFVIIIFc might be a resultant of theinteraction of the Fc with the Fcγ receptors which is abolished by theFcγ non-binding mutant.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

TABLE 1 Polynucleotide Sequences A. B-Domain Deleted FVIIIFc(i) B-Domain Deleted FVIIIFc Chain DNA Sequence (FVIII signal peptide underlined, Fc region in bold)(SEQ ID NO: 1, which encodes SEQ ID NO: 2)  661                A TGCAAATAGA GCTCTCCACC TGCTTCTTTC  721TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC  781TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC  841CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG  901TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC  961TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG 1021CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG 1081CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG 1141GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC 1201TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA 1381CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC 1441ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG 1741AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG 1801ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 2461ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 2521AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 3241GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 3301CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG 4261AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA 4321CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041GCGAGGCACA GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC 5101TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 5161CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA 5221AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG 5281AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC 5341TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 5401AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT 5461CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC 5521CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA 5581CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA 5641AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA 5701ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A(ii) Fc DNA sequence (mouse Igκ signal peptide underlined) (SEQ ID NO: 3, which encodes SEQ ID NO: 4) 7981                         ATGGA GACAGACACA 8041CTCCTGCTAT GGGTACTGCT GCTCTGGGTT CCAGGTTCCA CTGGTGACAA AACTCACACA 8101TGCCCACCGT GCCCAGCACC TGAACTCCTG GGAGGACCGT CAGTCTTCCT CTTCCCCCCA 8161AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 8221GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT 8281AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC 8341CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 8401AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 8461CCACAGGTGT ACACCCTGCC CCCATCCCGC GATGAGCTGA CCAAGAACCA GGTCAGCCTG 8521ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG 8581CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC 8641CTCTACAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC 8701TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 8761GGTAAA B. Full Length FVIIIFc(i) Full Length FVIIIFc DNA Sequence (FVIII signal peptide underlined, Fc region in bold)  (SEQ ID NO: 5, which encodes SEQ ID NO: 6)  661                    ATG CAAATAGAGC TCTCCACCTG  721CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC  781AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG  841ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC  901TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT  961GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA 1021GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC 1081TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT 1141CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC 1201TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA 1261TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC 1321ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA 1381CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC 1441TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA 1501CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT 1561ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTG 1621GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT 1681TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG 1741TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA 1801TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT 1861CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA 1921AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG 1981TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT 2041GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT 2101GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC 2161AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG 2221GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT 2281CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG 2401CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA 2521GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA 2581AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT 2641TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC 2881TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA 2941CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC 3001TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC 3061TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA 3241CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT 3301TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC 3361AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT 3421TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA 3601ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG 3661TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC 3721CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG 3841GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC 3961TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC 4021ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT 4081ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA 4321TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT 4381AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT 4441ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA 4501AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA 4681ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA 4741AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC 4801GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA 5041AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC 5101CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC 5161AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT 5401AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA 5461CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA 5521AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA 5641AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC 5761AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG 5821CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA 5881TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG 6001TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA 6061TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT 6121TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC 6181TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA 6241GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC 6301AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG 6361ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG 6421GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA 6481TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT 6541ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG 6601CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA 6661GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT 6721ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG 6781GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC 6841TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA 6901GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC 6961TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC 7021CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG 7081GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA 7141TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT 7201CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG 7261TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA 7321GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG 7381ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG 7441GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA 7501ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA 7561TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC 7621CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA 7681CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA 7741GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG 7801ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 7861TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 7921GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA 7981CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 8041GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 8101CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 8161GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 8221CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 8281GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG 8341GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 8401GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A(ii) Fc (same sequence as A (ii) (SEQ ID NO: 3))]

TABLE 2 Polypeptide SequencesA. B-Domain Deleted FVIII-Fc Monomer Hybrid (BDD FVIIIFc monomer dimer): created by coexpressing BDD FVIIIFc and Fc chains. Construct =HC-LC-Fc fusion. An Fc expression cassette is cotransfected with BDDFVIII-Fc to generate the BDD FVIIIFc monomer-. For the BDD FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; remaining B domain sequence is shown in italics. Signal peptides are  underlined.i) B domain deleted FVIII-Fc chain (19 amino acid signal sequence underlined)  (SEQ ID NO: 2) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKii) Fc chain (20 amino acid heterologous  signal peptide from mouse Igκchain   underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKB. Full length FVIIIFc monomer hybrid (Full length FVIIIFc monomer dimer): created by coexpressing FVIIIFc and Fc chains. Construct =HC-B-LC-Fc fusion. An Fc  expression cassette is cotransfected with full length FVIII-Fc to generate the full length FVIIIFc monomer. For the FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; B domain sequence is shown in italics. Signal peptides  are underlined.i) Full length FVIIIFc chain (FVIII signal   peptide underlined(SEQ ID NO: 6) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFENQASRPYNIYPHGITDVRPLYSRRLPEGVEHLEDFPILPGEIFEYEWTVTVEDGPTESDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDERNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFEHEMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLEVSSCDENTGDYYEDSYEDISAYLL SENNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRENFVEPNETETYFWEVQHHMAPTEDEFDCKAWAYFSDVDLEEDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETESWYFTENMERNCRAPCNIQMEDPTFEENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVREKEEYEMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNECQTPLGMASGHIRDFQITASGQYGQWAPELARLHYSGSINAWSTKEPFSWIEVDLLAPMIIHGIETQGARQEFSSLYISQFIIMYSLDGEKWQTYRGNSTGTLMVFFGNVDSSGIEHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSEARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMEVTGVTTQGVESLLTSMYVEEFLISSSQDGHQWTLFFQNGEVEVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGKii) Fc chain (20 amino acid heterologous  signal peptide from mouse Igκchain   underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

What is claimed is:
 1. A method of reducing an inhibitory Factor VIII(FVIII) immune response in a subject having hemophilia A, comprisingadministering to the subject a chimeric polypeptide comprising a FVIIIpolypeptide and an Fc, wherein the subject has developed an inhibitoryimmune response to a FVIII protein.
 2. The method of claim 1, furthercomprising measuring the level of the inhibitory FVIII immune responsebefore the administration.
 3. The method of claim 2, further comprisingcomparing the level of the inhibitory FVIII immune response after theadministration with the level of the inhibitory FVIII immune responsebefore the administration.
 4. The method of claim 1, wherein theinhibitory FVIII immune response comprises inhibitory antibodies to theFVIII protein, a cell-mediated immune response, or one or more clinicalsymptoms selected from increased bleeding tendency, high factor VIIIconsumption, lack of response to a therapy comprising administration ofthe FVIII protein, decreased efficacy of a therapy comprisingadministration of the FVIII protein, and shortened half-life of theFVIII protein.
 5. The method of claim 1, wherein the inhibitory FVIIIimmune response comprises an inhibitory antibody to the FVIII protein.6. The method of claim 4, wherein the concentration of the inhibitoryantibodies prior to the administration is at least about 0.6 BethesdaUnits (BU).
 7. The method of claim 4, wherein the concentration of theinhibitory antibodies prior to the administration is at least about 1.0BU.
 8. The method of claim 4, wherein the concentration of theinhibitory antibodies after the administration is less than about 1.0BU.
 9. The method of claim 4, wherein the concentration of theinhibitory antibodies after the administration is less than about 0.6BU.
 10. The method of claim 1, which (a) reduces the number ofanti-FVIII antibodies in the subject compared to the number prior toadministration of the chimeric polypeptide; and/or (b) reduces the titerof anti-FVIII antibodies in the subject compared to the titer prior toadministration of the chimeric polypeptide.
 11. The method of claim 1,wherein the FVIII polypeptide comprises human FVIII having a full orpartial deletion of the B domain.
 12. The method of claim 11, whereinthe B domain-deleted FVIII is a single chain FVIII.
 13. The method ofclaim 1, wherein the FVIII polypeptide comprises an amino acid sequenceat least about 90% identical to SEQ ID NO:2.
 14. The method of claim 1,wherein the chimeric polypeptide comprises a monomer dimer hybridcomprising a first polypeptide comprising the FVIII polypeptide and theFc and a second polypeptide consisting of an Fc.
 15. The method of claim1, further comprising administering the chimeric polypeptide foron-demand treatment.
 16. The method of claim 1, further comprisingadministering the chimeric polypeptide for prophylactic treatment. 17.The method of claim 16, wherein the chimeric polypeptide is administeredat an effective dose between 25 IU/kg and 65 IU/kg.
 18. The method ofclaim 17, wherein the chimeric polypeptide is administered at a dosinginterval of three to seven days.
 19. The method of claim 1, wherein thechimeric polypeptide comprises one or more half-life extending moietiesin addition to the Fc.
 20. The method of claim 1, wherein the chimericpolypeptide is administered intravenously.
 21. The method of claim 4,wherein the concentration of the inhibitory antibodies prior to theadministration is at least about 5.0 Bethesda Units (BU).
 22. The methodof claim 7, wherein the concentration of the inhibitory antibodies afterthe administration is less than about 0.6 BU.
 23. The method of claim21, wherein the concentration of the inhibitory antibodies after theadministration is less than about 0.6 BU.
 24. A method of reducing aninhibitory FVIII immune response in a subject having hemophilia A,comprising administering to the subject a chimeric polypeptidecomprising a FVIII polypeptide and an Fc, wherein the subject hasdeveloped inhibitory antibodies to full-length rFVIII or B domaindeleted rFVIII, wherein the concentration of the inhibitory antibodiesprior to the administration is at least about 1.0 BU and wherein theconcentration of the inhibitory antibodies after the administration isless than about 0.6 BU.
 25. A method of reducing an inhibitory FVIIIimmune response in a subject having hemophilia A, comprisingadministering to the subject a chimeric polypeptide comprising a FVIIIpolypeptide and an Fc, wherein the subject has developed inhibitoryantibodies to full-length rFVIII or B domain deleted rFVIII, wherein theconcentration of the inhibitory antibodies prior to the administrationis at least about 5.0 BU and wherein the concentration of the inhibitoryantibodies after the administration is less than about 0.6 BU.